• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    多位點序列分型(MLST)在艾伯特埃希菌鑒定中的應(yīng)用

    2015-02-14 08:08:56許彥梅鄧建平孫松松熊衍文
    中國人獸共患病學(xué)報 2015年11期
    關(guān)鍵詞:艾伯特株菌埃希菌

    劉 祥,許彥梅,王 斌,鄧建平,肖 波,孫松松,周 陽,熊衍文,王 紅

    多位點序列分型(MLST)在艾伯特埃希菌鑒定中的應(yīng)用

    劉 祥1,許彥梅2,王 斌1,鄧建平1,肖 波1,孫松松1,周 陽1,熊衍文2,王 紅1

    目的 探討多位點序列分型技術(shù)在新病原艾伯特埃希菌發(fā)現(xiàn)與鑒定中的應(yīng)用。方法 采用MLST技術(shù)對生鮮肉中分離的30株疑似艾伯特埃希菌的7個管家基因進行PCR擴增、測序、DNAstar軟件分析,核酸序列上傳至大腸桿菌MLST數(shù)據(jù)庫進行比對,確定其等位基因編號及基因序列型(ST型),并利用MEGA6.0軟件Neighbor-joining法構(gòu)建遺傳進化樹,與大腸埃希菌、志賀氏菌、艾伯特埃希菌和傷寒沙門氏菌參考菌株進行親緣性分析。結(jié)果 30株菌可分為7種新序列型(16株)和4種已知序列型(14株),N-J分析顯示與艾伯特埃希菌參考菌株具有高度親緣性,而與大腸埃希菌、志賀氏菌、弗格森埃希菌和鼠傷寒沙門氏菌存在較大差異。結(jié)論 MLST在管家基因水平上準確地確定艾伯特埃希菌的種屬關(guān)系,首次在國內(nèi)發(fā)現(xiàn)艾伯特埃希菌的存在。

    多位點序列分型技術(shù)(MLST);艾伯特埃希菌;新病原

    艾伯特埃希菌是一種新發(fā)現(xiàn)的致瀉性病原菌,它可引起人類的散發(fā)感染和食物中毒[1-7]。該菌最初從孟加拉腹瀉兒童的糞便中分離,被鑒定為蜂房哈夫尼菌[1],通過生化表型特征、16S rDNA序列和DNA雜交分析,Huys等將此類蜂房哈夫尼菌命名為埃希氏菌屬種的一個新種——艾伯特埃希菌(Escherichiaalbertii)[8]。由于缺乏艾伯特埃希菌的生化特性,迄今仍無該菌種的商品化鑒定系統(tǒng)。常規(guī)的細菌鑒定系統(tǒng)經(jīng)常將艾伯特埃希菌鑒定為哈夫尼菌、沙門氏菌、魯氏耶爾森菌,最常見的確定為大腸埃希菌。缺乏動力、不發(fā)酵木糖、不發(fā)酵乳糖和產(chǎn)生β-D葡萄糖醛酸酶被認為是艾伯特埃希菌常見生化特性,并且攜帶有編碼緊密粘附素(Intimin)基因eae,此外菌株還通常攜帶編碼細胞腫脹壞死毒素(cytolethal distending toxin,CDT)基因cdtABC[3,13]。

    多位點序列分型(MLST)通過對7個管家基因核酸序列的分析,將每個基因的序列與MLST數(shù)據(jù)庫比對,得到相應(yīng)的等位基因編號,按照指定排列順序形成ST型。ST型可用于國際實驗室間比對、溯源以及分子流行病學(xué)、遺傳進化等研究;同時根據(jù)等位基因圖譜使用配對差異矩陣方法構(gòu)建系統(tǒng)進化樹,與參考菌株的相應(yīng)序列進行親緣性分析,從而準確地研究菌株遺傳進化關(guān)系,以確定種屬[9-11]。通過MLST分析,艾伯特埃希菌明顯不同于腸桿菌科及埃希菌屬中的其它種[3-8]。

    本研究對四川省自貢市農(nóng)貿(mào)市場生鮮肉食品中分離出的30株疑似艾伯特埃希菌進行多位點序列分型,通過與NCBI數(shù)據(jù)庫收錄的大腸埃希菌、志賀氏菌、艾伯特埃希菌和傷寒沙門氏菌的相應(yīng)序列進行親緣性分析,為確定艾伯特埃希菌提供依據(jù)。

    1 材料與方法

    1.1 菌株來源與鑒定 從2013年3月-2014年7月,在中國四川省自貢市的超市和農(nóng)貿(mào)市場共收集446份生肉,包含92份牛肉、41份豬肉、22份羊肉、53份雞肉、189份雞腸、21份鴨肉、28份鴨腸。根據(jù)艾伯特埃希菌相關(guān)研究資料,取生肉樣品25 g,接種225 mL EC,20 ℃增菌24~36 h,取增菌液進行eae基因PCR檢測[3]。eae陽性增菌液接種麥康凱瓊脂平板,36 ℃培養(yǎng)過夜,取不發(fā)酵乳糖的白色菌落進行eae基因檢測。最后分離eae陽性且不發(fā)酵乳糖、不發(fā)酵木糖、動力陰性、cdtB基因陽性的疑似艾伯特埃希菌30株。

    1.2 儀器與試劑

    (1)主要儀器:PCR儀(SensoQuest Labcycler)、Bio-Rad凝膠成像系統(tǒng)(GEL DocXR+)。

    (2)主要試劑:2×Taq MasterMix(含染料)、DNA Marker(DM1000)、去離子水:購自康為世紀公司;核酸染料(GeneGreen):購自天根生化科技(北京)有限公司;PCR引物合成及測序:由生工生物工程(上海)有限公司完成。

    1.3 DNA模板制備及反應(yīng)體系 挑取少許疑似艾伯特埃希菌純培養(yǎng)物接種至EC營養(yǎng)肉湯,36 ℃培養(yǎng)18~24 h。吸取1 mL菌懸液于1.5 mL無菌離心管,8 000 r/min,離心10 min,用500 μL無菌去離子水重懸并煮沸10 min,將重懸液10 000 r/min離心5 min,取上清液作為PCR模板。PCR體積為50 μL,2×Taq MasterMix 25 μL,ddH2O 20μL,上、下游引物(10 μmol/L)各2 μL,模板DNA1 μL。

    1.4 MLST引物及反應(yīng)程序 管家基因位點選擇參照大腸桿菌MLST數(shù)據(jù)庫(http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/primersColi_html)提供的分型方案,PCR引物序列、退火溫度及產(chǎn)物大小詳見參考文獻[12]。反應(yīng)程序為94℃ 5min預(yù)變性,按照94 ℃ 30 s、退火30 s、72 ℃ 45 s進行30個循環(huán),最后一個循環(huán)結(jié)束后72 ℃延伸7 min。每次擴增均以無DNA模板的體系作為空白對照。

    1.5 結(jié)果觀察 取PCR產(chǎn)物5 μL以1.5%瓊脂糖凝膠(含1/10 000核酸染料)進行電泳,通過凝膠成像系統(tǒng)觀察是否擴增出目的大小片段,將PCR產(chǎn)物送至上海生工生物工程公司雙向測序。

    1.6 數(shù)據(jù)分析 利用SeqMan軟件對PCR產(chǎn)物序列與數(shù)據(jù)庫中的管家基因標準序列進行拼接和校正,校正后的7個管家基因序列同時上傳至E.coliMLST數(shù)據(jù)庫,確定菌株的等位基因型和序列類型。并根據(jù)Ooka等人[3]描述,基于7個管家基因的串聯(lián)序列,利用MEGA6.0軟件Neighbor-joining法構(gòu)建系統(tǒng)進化樹,與國際數(shù)據(jù)庫中已收錄的大腸桿菌、志賀氏菌、艾伯特埃希菌和傷寒沙門氏菌進行親緣性比對,探究該30株菌的種屬關(guān)系。

    2 結(jié) 果

    2.1 MLST分型結(jié)果 通過不斷優(yōu)化擴增條件、選取引物,所有菌株的7個管家基因序列均得到雙向峰圖完好的序列,與MLST數(shù)據(jù)庫中的等位基因剪切比對,結(jié)果顯示14株菌分別為4種已知ST型,而其余16株菌的各等位基因雙向序列通過互聯(lián)網(wǎng)上傳至數(shù)據(jù)庫,被接受并確認為7種新ST型,見表1。

    2.2 MLST聚類圖譜分析 根據(jù)7個管家基因首尾串聯(lián)整合成的3 423 bp核苷酸序列圖譜分析,表明研究中的30株菌與大腸埃希菌、志賀氏菌、弗格森大腸埃希菌、鼠傷寒沙門氏菌存在較大差異。

    表1 30株菌MLST型別Tab.1 MLST STs of 30 strains

    30株艾伯特埃希菌中鑒別出11種序列型,均與艾伯特埃希菌LMG20976、KF1具有高度親緣關(guān)系,其中SP140602和SP140701兩株菌的序列型與艾伯特埃希菌KF1一致,見圖1。結(jié)果表明30株菌全部鑒定為艾伯特埃希菌。

    圖1 30株菌與大腸埃希菌、弗格森埃希菌、艾伯特埃希菌、志賀氏菌、傷寒沙門氏菌系統(tǒng)發(fā)育關(guān)系分析結(jié)果

    Fig.1 Phylogenetic relationships of 30 strains withE.coli,E.fergusonii,E.albertii,Shigellaspp. andSalmonellaentericaserovar Typhi

    2.3 GenBank核酸序列索取號 本研究中30株艾伯特埃希菌的7個管家基因已全部上傳至GenBank數(shù)據(jù)庫,登陸號為:KP015856-KP016011、KP064411-KP064472。

    3 討 論

    艾伯特埃希菌是一種新發(fā)現(xiàn)的腸道致病菌。起初通過常規(guī)檢測方法被鑒定為致病性大腸桿菌(EPEC)或者出血性大腸桿菌(EHEC),對該菌的誤診漏診容易造成公眾健康潛在的危險,也會影響臨床治療效果。由于目前對其研究較少,仍沒有標準化檢測方法和商品化鑒定系統(tǒng),利用分子生物學(xué)技術(shù)在基因水平上研究遺傳進化關(guān)系,則成為菌種鑒定的關(guān)鍵。

    利用MLST對30株疑似艾伯特埃希菌分型過程中,存在個別菌株的管家基因序列在大腸桿菌MLST數(shù)據(jù)庫中未找到完全匹配的等位基因型,可能屬于新型別。采用雙向測序,將其完好的序列和峰圖文件上傳至MLST數(shù)據(jù)庫以待確認,上傳前需核查PCR產(chǎn)物序列和峰圖文件,避免誤判等位基因型及新型別;此外,在本實驗樣品7個管家基因PCR擴增時,除purA外的其余6個管家基因產(chǎn)物測序峰圖基本為單峰,個別出現(xiàn)重峰、雜峰的通過多次擴增和降低模板濃度得到改善;而全部樣品purA基因測序產(chǎn)物重峰、雜峰情況嚴重,存在非特異產(chǎn)物干擾,結(jié)果無法用于比對分析。利用提高退火溫度,降低模板濃度,多次擴增后測序效果并不理想,最終改用數(shù)據(jù)庫中purA其它引物進行擴增后,產(chǎn)物特異性明顯提高,正反向測序峰圖完好。這也提示我們發(fā)現(xiàn)測序峰圖不完好時,可通過多次PCR擴增測序、優(yōu)化擴增條件和選取不同引物進行解決。

    傳統(tǒng)檢測方法在新病原菌鑒定中存在局限,而MLST可通過研究管家基因圖譜與參考菌株相應(yīng)序列構(gòu)建系統(tǒng)進化樹,根據(jù)聚類分析結(jié)果判斷菌種歸屬,在新病原菌的鑒定和常見細菌新的變異方面發(fā)揮積極的作用。本研究MLST聚類分析結(jié)果顯示30株菌為艾伯特埃希菌,屬國內(nèi)首次發(fā)現(xiàn)并證實艾伯特埃希菌的存在。然后MLST針對的7個管家基因是大腸埃希菌的保守基因,如若標本中混有大腸桿菌,PCR擴增為混合產(chǎn)物無法區(qū)分,故不能直接作為該細菌PCR快速診斷。目前擬針對艾伯特埃希菌的特異基因建立PCR快速診斷方法。

    綜上所述,MLST為最終確認艾伯特埃希菌提供依據(jù)。同時也適用于其他新病原菌的診斷以及后續(xù)的分子流行病學(xué)研究、菌群遺傳多態(tài)性、溯源等,為制定有效的監(jiān)測體系和預(yù)防疾病的流行與暴發(fā)提供有力的技術(shù)支持。

    [1]Albert MJ, Alam K, Islam M, et al.Hafniaalvei, a probable cause of diarrhea in humans[J]. Infect Immun, 1991, 59: 1507-1513.

    [2]Konno T, Yatsuyanagi J, Takahashi S, et al. Isolation and identification ofEscherichiaalbertiifrom a patient in an outbreak of gastroenteritis[J]. Jap J Infect Dis, 2012, 65: 203-207. DOI: 10.7883/yoken.65.203

    [3]Ooka T, Seto K, Kawano K, et al. Clinical significance ofEscherichiaalbertii[J]. Emerg Infect Dis, 2012, 18: 488-492. DOI: 10.3201/eid1803.111401

    [4]Asoshima N, Matsuda M, Shigemura K, et al. Identification ofEscherichiaalbertiias a causative agent of a food-borne outbreak occurred in 2003[J]. Jap J Infect Dis, 2014, 67: 139-140.

    [5]Murakami K, Etoh Y, Tanaka E, et al. Shiga toxin 2f-producingEscherichiaalbertiifrom a symptomatic human[J]. Jap J Infect Dis, 2014, 67: 204-208.

    [6]Oaks JL, Besser TE, Walk ST, et al.Escherichiaalbertiiin wild and domestic birds[J]. Emerg Infect Dis, 2010, 16: 638-646. DOI: 10.3201/eid1604.090695

    [7]Ooka T, Tokuoka E, Furukawa M, et al. Human gastroenteritis outbreak associated withEscherichiaalbertii, Japan[J]. Emerg Infect Dis, 2013, 19: 144-146. DOI: 10.3201/eid1901.120646

    [8]Huys G, Cnockaert M, Janda JM, et al.Escherichiaalbertiisp. nov., a diarrhoeagenic species isolated from stool specimens ofBangladeshichildren[J]. Int J Systemat Evolutionary Microbiol, 2003, 53: 807-810.

    [9]Ji XW, Liao YL, Mao XH, et al. The research progress of MLST analysis in the application of Pathogenic microorganisms genotyping[J]. Int J Lab Med, 2011, 32(2): 246-247. DOI: 10.3969/j.issn.1673-4130.2011.02.051 (in Chinese) 姬小薇,廖亞玲,毛旭虎,等. MLST分析在病原微生物基因分型應(yīng)用中的研究進展[J].國際檢驗醫(yī)學(xué)雜志,2011,32(2):246-247.

    [10]Zuo TT, Li YW, Han XL, et al. Two new sequence type isolates ofBacillusanthracisby multilocus sequence typing[J]. Acta Microbiologica Sinica, 2012, 52(1): 120-123. (in Chinese) 左庭婷,李巖偉,韓雪蓮,等. 兩株炭疽芽胞桿菌 MLST 新序列型(ST) [J].微生物學(xué)報,2012,52(1):120-123.

    [11]Deng XL, Guan DW, Li W, et al. MLST technique applied to molecular epidemiological research on neisseria meningitides isolates[J]. South China J Prev Med, 2008, 34(3): 10-14. (in Chinese) 鄧小玲,管大偉,黎薇,等. MLST分型技術(shù)應(yīng)用于腦膜炎奈瑟菌的分子流行病學(xué)研究[J]. 華南預(yù)防醫(yī)學(xué),2008,34(3):10-14.

    [12]Bai XN, Zhao AL, Xia SL, et al. Multilocus sequence typing of non-O157 Shiga toxin-producingEscherichiacoliisolates[J]. Chin J Zoonoses, 2012, 28(6): 544-548. (in Chinese) 白向?qū)帲w愛蘭,夏勝利,等. 非O157產(chǎn)志賀毒素大腸桿菌分離株的多位點序列分型研究[J]. 中國人獸共患病學(xué)報,2012,28(6): 544-548.

    [13]Bai XN, Wang H. The research progress onEscherichiaalbertii[J]. Chin J Zoonoses, 2014, 34(2): 154-158. DOI: 10.3760/cma.j.issn.0254-5101.2014.02.016 (in Chinese) 白向?qū)?,王紅. 艾伯特埃希菌研究進展[J]. 中華微生物學(xué)和免疫學(xué)雜志,2014,34(2): 154-158.

    DOI:10.3969/j.issn.1002-2694.2015.11.010

    Abstract:MicroRNAs (miRNAs) are key regulators of gene expression. The miR-36 family has only been authentically found in helminth includingAscarisspp.,Caenorhabditiselegans,Brugiamalayi, andSchistosomamansoni. And the family member named miR-36f was identified to exist only in the egg and larval stage ofAscarissuum(A.suum) in our previous study.We herein preliminarily evaluated the function of miR-36f via its mimics and inhibitors by usingA.suumas model organism. The miR-36f mimics, inhibitors, mimics negative controls and inhibitor negative controls were absorbed by the different groups larvae via soaking method. Three days later, the mice were infected with the larvae above. And the larvae were recovered from the liver and lung of the mice respectively. The larvae of each group were counted and their length and width were measured, and the two target genes of miRNA were analyzed by qRT-PCR.The results showed that development and infective numbers of theA.suuminfective larvae were significantly influenced by miR-36f inhibitors (P<0.05), when compared with the mimics (P>0.05).The expression of the two key target genes of miR-36f named OST48 and cytochrome b were similar to that of inhibitor and mimics. When them iR-36f was influenced by its inhibitor, the development and infective ability ofA.suumlarvae were reduced, which indicated that the miR-36f might be associated with development and infectivity of theA.suumlarvae. Furthermore, miR-36f inhibitors seemed more effective than its mimics. The results would be helpful for better understanding and novel control strategy for parasitic helminths.

    Keywords:Ascarissuum; asu-miR-36f; infectivity; development

    Supported by the Science Fundfor Creative Research Groups of Gansu Province (Grant No.1210RJIA006)

    Corresponding author: Xu Min-jun, Email:xuminjun@caas.cn

    MicroRNAs (miRNAs) are small non-coding RNAs with increasingly recognized regulating roles played in gene expression in animals, plants and parasites by binding to mRNAs in 3′-untranslated regions (3′-UTR), and also in coding domain sequences or 5′-UTR[1-4]. And up to now, members of the miR-36 family have been only authenticated in helminths, includingAscarisspp.[5],Caenorhabditiselegans[6],Brugiamalayi[7],Schmidteamediterranea[8], andSchistosomamansoni[9]. The miR-36 family members were highly conserved, stayed in cluster in different organisms, and adult- and larva-specifically expressed; knocking down the miR-36 cluster will lead to embryonic and larval deadliness[10-11]. These reports indicated that the miR-36 members play vital roles in worms. Besides, the main control measure for the epidemic of worms at present is anthelmintic drugs, which easily results in the drug resistance of the parasite and drug residues in animal products[12-14]. Therefore, alternative controlling measure is urgently needed for the replacement of anthelmintic drugs for animal welfare and food safety, and changing miRNA expression abundance with convenient way is likely to be a new way to prevent and control the parasites.

    A.suumis a globally parasitic nematode which causes infection in pigs and human with high prevalence rates, especially in developing countries[15-17], and the identification of its genome, proteome, and transcriptome made the parasite be a fine model[18-20]. We herein usedA.suumas a model to evaluate the roles of one miR-36 member named miR-36f, which is specifically expressed in the egg and larval stage ofA.suum. Furthermore, two key target genes of miR-36f, named OST48 (gi|171279896) and cytochrome b (gi|76250725), were detected with qRT-PCR after the over- and down-regulation of miR-36f with the corresponding mimics and inhibitors.

    Materials and methods

    Larvae preparation

    A.suumembryos were collected from the uteri of live adult females, which were obtained from slaughtered pigs in Lanzhou, Gansu Province of China. The embryos were incubated in a culture dish at 28 ℃ for 30 days to allow embryonic development. After that, eggshells were removed with 7.5% v/v sodium hypochlorite overnight at 37 ℃ and shaking with glass beads[21]. A total of 1.0×105larvae were obtained and cultured in DMEM (HyClone) containing 50% calf serum, penicillin G potassium (1 000 units/mL), amphotericin B (10 μg/mL), and streptomycin sulfate (1 mg/mL). The mixture was then evenly spread into a dish of 5 holes, and the miRNA mimics, inhibitors, mimics negative controls and inhibitor negative controls were added to the 4 holes respectively, with the last one left as blank control. The experimental nematodes were cultured at 37 ℃ with 5% CO2for 72 h following gently hand shook 2-3 times each day.

    Animals

    Fifty 8-week-old BALB/c mice of specific-pathogen-free (SPF) grade were purchased from Lanzhou Veterinary Research Institute Animal Center. The mice were equally divided into five groups, housed in sterile cages, and fed with pelleted food and wateradlibitum. And the mice were acclimatized to these conditions for one week before the experiment. Animal experiments were performed strictly according to the institutional guidelines for animal ethics of China.

    Preparation of miRNA inhibitors, mimics and negative controls

    The miRNA inhibitors, mimics, and their negative controls (Table 1) were obtained from Invitrogen (Shanghai) and prepared a final concentration of 20 μM by following the instructions respectively. Nucleotides of miRNA inhibitor and its control were modified with 2’-O-methyl.

    Challenge infection and measurement of larvae

    After soaking for 72 h, larvae of the 5 groups were collected separately. And the 5 groups BALB/c

    Tab.1 Sequences of miRNA inhibitor, mimic and their negative controls

    Note:aN.C indicates negative control.

    mice were subjected to be orally infected with about 5 000 larvae for each. Baermann method was adopted to recover the larvae that migrated to the livers and lungs 4 days later[22]. In a nutshell, livers and lungs of the experimental and the control mice were collected and carefully ground with a mortar, and transferred to a small gauze bag containing 30 mL sterile physiological saline with kanamycin (100 mg/L) and ampicillin (100 mg/L) added, and incubated at 37 ℃ for 24 hours. The larvae recovered were counted and measured with a microscope having micrometer (Olympus) with 20 worms detected for each.

    qRT-PCR of target genes after miRNA interventions

    Two target genes named OST48 and cytochrome b were then quantified by qRT-PCR following the over-and/or down-regulation of miR-36f. Briefly, total RNA from the transfectedA.suumlarvae were extracted with Trizol reagent (Invitrogen) in accordance with the manufacture’s protocol. The purity of total RNA was confirmed by BioPhotometer plus (Eppendorf). Reverse transcription to obtain cDNA by using EasyScript First-Strand cDNA Synthesis SuperMix (Transgene, Beijing). Real-time quantitative PCR was conducted using an ABI PRISM?7500 Sequence Detection System. All of the primers (Table 2) were synthesized by Shenggong Co, Ltd., China. The actin gene (EU109284) was used as the endogenous control. The amplification cycle conditions were performed as follows: 95 ℃ for 5 min, followed by 40 cycles of 95 ℃ for 15 s, 65 ℃ for 15 s, and 72 ℃ for 32 s. The relative quantification of each miRNA and its target gene was calculated using the equation: N = 2-ΔCt, ΔCt = CtmiRNA-Ctactin[23]. All reactions were conductedin triplicate.

    Tab.2 Sequences of primers used in the amplification of target and endogenous control genes

    PrimerSequence(5′to3′)cytochromeb?FAGTTTTGGGGGTTGTGTCTTTGcytochromeb?RACACTGCCCAAGTCAACTCAAAOST48?FAAAAGTTGGAGTTTGAGGAGCAOST48?RATCCACAAGCACAAGAATACGAβ?actin?FCTCGAAACAAGAATACGATGβ?actin?RACATGTGCCGTTGTATGATG

    Results and discussion

    There was a significant difference between the inhibitor treated group and the control, including the larvae recovered number, body length and body width (P<0.05). For larvae recovered number, there was 42.5±23.72 larvae recovered from the inhibitor treated groups (P<0.05), while it was 69.44±34.86, 55.77±27.30, 67.86±27.82, 75.00±33.07 recovered from the mimics groups and other 3 control groups, respectively (Figure 1). For body length, it measures 367±133.29 μm in average in the inhibitor group, while that of controls is 460.82±165.12 μm in average (P<0.05) (Figure 2A). For the body width, it is 17.04±6.37 μm and 23.44±6.90 μm respectively for the inhibitor treated groups and controls (P<0.05)(Figure 2B). For the immune protection of the

    host, most of the invasion larvae were killed with only those worms strongly enough to survive. Therefore, although 5 000 larvae were orally infected, only a small number of larvae were successfully recovered, similar to the report by Islam et al[24]. No significant differences were found (P>0.05) in mimics, which was similar to the study reported by Puthiyakunnon[25].This phenomenon might suggest that, for its specific and crucial regulating functions for the development and infectivity of larvae, miR-36f was also critically controlled by organism, and any changes over the saturation level will not obtain functional enhancement.

    Data are expressed as the mean values±standard deviations in each group. The symbol"*" indicatesP<0.05.

    Fig.1 Number of recoveredA.suumlarvae from the five experimental groups

    Data are expressed as the mean values±standard deviations in each group. The symbol"*" indicates P<0.05.Fig.2 Mean body length (A) and width (B) of recovered larvae from the five experimental groups

    The expression of OST48 genes was significantly up-regulated after inhibitor treatment(P<0.05), and only slightly decreased after mimics treated (Figure 3A). In contrast to cytochrome b, OST48 was significantly reduced with the mimics (P<0.01), and only slightly increased after inhibitors treatment (P>0.05) (Figure 3B). The two target genes were obtained via enrichment analysis from a total of 216 target genes of miR-36f in our previous study, indicating essential roles of the two genes in the regulating network of miR-36f for the organisms.OST48 is an important part of the oligosaccharyl transferase which plays an important role in N-linked glycosylation[26-27]. And cytochrome b is involved in the oxidative phosphorylation, and is important for cellular metabolism[28-30]. When treated with mimics or inhibitors,miR-36f of the organism was influenced, leading to a changed regulation and metabolism balance, and the expression changes of targets. The phenomenon indicated that miR-36f did have functions of gene regulation in the worms. Besides, it was found that the two targets were not changed as similar trends, which confirmed the previous conclusion that cellular pathways might be regulated with one or more miRNAs[31].

    A: Quantification of OST48. B: Quantification of cytochrome b. The symbol"*" indicates P<0.05; "**" indicates P<0.01.

    Fig.3 Target gene relative expression analysis of groups interfered with mimics, inhibitors, negative control, inhibitor negative control and normal saline by qRT-PCR

    Conclusions

    By usingA.suumas model organism, we identified that the egg and larva stage-specific miRNA, miR-36f, involves in the infectivity and development of the parasite, which would help for better understanding and novel control strategy development of parasitic helminths.

    References

    [1]Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2): 281-297.

    [2]Lai EC. MicroRNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation[J]. Nat Genet, 2002, 30(4): 363-364.

    [3]Ivashchenko AT, IssabekovaAS, Berillo OA. MiR-1279, miR-548j, miR-548m, and miR-548d-5p binding sites in CDSs of paralogous and orthologous PTPN12, MSH6, and ZEB1 genes[J]. Biomed Res Int, 2013, 2013: 902467. DOI: 10.1155/2013/902467

    [4]da Sacco L, Masotti A. Recent insights and novel bioinformatics tools to understand the role of MicroRNAs binding to 5’untranslated region[J]. Int J Mol Sci, 2012, 14(1): 480-495. DOI: 10.3390/ijms14010480

    [5]Wang J, Czech B, Crunk A, et al. Deep small RNA sequencing from the nematodeAscarisreveals conservation, functional diversification, and novel developmental profiles[J]. Genome Res, 2011, 21(9): 1462-1477. DOI: 10.1101/gr.121426.111

    [6]de Wit E, Linsen SE, Cuppen E, et al. Repertoire and evolution of miRNA genes in four divergent nematode species[J].Genome Res, 2009, 19(11):2064-2074. DOI: 10.1101/gr.093781.109

    [7]Poole CB, Davis PJ, Jin J, et al. Cloning and bioinformatic identification of small RNAs in the filarial nematode,Brugiamalayi[J]. Mol Biochem Parasitol, 2010, 169(2): 87-94. DOI: 10.1016/j.molbiopara.2009.10.004

    [8]Palakodeti D, Smielewska M, Graveley BR. MicroRNAs from the PlanarianSchmidteamediterranea: a model system for stem cell biology[J]. RNA, 2006, 12(9): 1640-1649.

    [9]Marco A, Kozomara A, Hui JH, et al. Sex-biased expression of microRNAs inSchistosomamansoni[J]. PLoS Negl Trop Dis, 2013, 7(9): e2402. DOI: 10.1371/journal.pntd.0002402

    [10]Miska EA, Alvarez-Saavedra E, Abbott AL, et al. MostCaenorhabditiselegansmicroRNAs are individually not essential for development or viability[J]. PLoS Genet, 2007, 3(12): e215.

    [11]Alvarez-Saavedra E, Horvitz HR. Many families ofC.elegansmicroRNAs are not essential for development or viability[J]. CurrBiol, 2010, 20(4): 367-373. DOI: 10.1016/j.cub.2009.12.051

    [12]Holm SA, Sorensen CR, Thamsborg SM, et al. Gastrointestinal nematodes and anthelmintic resistancein Danish goat herds[J]. Parasite, 2014, 21: 37. DOI: 10.1051/parasite/2014038

    [13]Martinez-Valladares M, Cordero-Perez C, Rojo-Vazquez FA. Efficacy of an antihelmintic combination in sheep infected withFasciolahepaticaresistant to albendazole and clorsulon[J]. ExpParasitol, 2014, 136: 59-62. DOI: 10.1016/j.exppara.2013.10.010

    [14]Urban JF Jr, Hu Y, Miller MM, et al.Bacillusthuringiensis-derived Cry5B has potent anthelmintic activity againstAscarissuum[J]. PLoS Negl Trop Dis,2013, 7(6): e2263. DOI:10.1371/journal.pntd.0002263

    [15]Buxton SK, Robertson AP, Martin RJ. Diethylcarbamazine increases activation of voltage-activated potassium (SLO-1) currents inAscarissuumand potentiates effects of emodepside[J]. PLoS Negl Trop Dis, 2014, 8(11): e3276. DOI: 10.1371/journal.pntd.0003276

    [16]Nejsum P, Parker ED Jr, Frydenberg J, et al. Ascariasis is a zoonosis in Denmark[J]. J ClinMicrobiol, 2005, 43(3):1142-1148.

    [17]Morimoto M, Zarlenga D, Beard H, et al.Ascarissuum: cDNA microarray analysis of 4th stage larvae (L4) during self-cure from the intestine[J]. Exp Parasitol, 2003, 104(3-4): 113-121.

    [18]Jex AR, Liu S, Li B, et al.Ascarissuumdraft genome[J]. Nature, 2011, 479(7374): 529-533. DOI: 10.1038/nature10553

    [19]Chehayeb JF, Robertson AP, Martin RJ, et al. Proteomic analysis of adultAscarissuumfluid compartments and secretory products[J]. PLoS Negl Trop Dis, 2014, 8(6): e2939. DOI: 10.1371/journal.pntd.0002939

    [20]Ma X, Zhu Y, Li C, et al. Comparative transcriptome sequencing of germline and somatic tissues of theAscarissuumgonad[J]. BMC Genomics, 2011, 12:481. DOI: 10.1186/1471-2164-12-481

    [21]Xu MJ, Chen N, Song HQ, et al.RNAi-mediated silencing of a novelAscarissuumgene expression in infective larvae[J]. Parasitol Res, 2010, 107(6): 1499-1503. DOI: 10.1007/s00436-010-2027-3

    [22]Chen N, Xu MJ, Nisbet AJ, et al.Ascarissuum: RNAi mediated silencing of enolase gene expression in infective larvae[J]. Exp Parasitol, 2011, 127(1): 142-146. DOI: 10.1016/j.exppara.2010.07.019

    [23]Sanchez-Freire V, Ebert AD, Kalisky T, et al. Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns[J]. Nat Protoc, 2012, 7(5): 829-838. DOI:10.1038/nprot.2012.021

    [24]Islam MK, Miyoshi T, Yamada M, et al. Pyrophosphatase of the roundwormAscarissuumplays an essential role in the worm’s molting and development[J]. Infect Immun. 2005, 73(4): 1995-2004.

    [25]Puthiyakunnon S, Yao Y, Li Y, et al. Functional characterization of three microRNAs of the Asian tiger mosquito,Aedesalbopictus[J]. Parasit Vectors, 2013, 6(1): 230. DOI: 10.1186/1756-3305-6-230

    [26]Mohorko E, Glockshuber R, Aebi M. Oligosaccharyltransferase: the central enzyme of N-linked protein glycosylation[J]. J Inherit Metab Dis, 2011, 34(4): 869-878. DOI:10.1007/s10545-011-9337-1

    [27]Roboti P, High S. The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 function as ubiquitous and selective modulators of mammalian N-glycosylation[J]. J Cell Sci,2012, 125(Pt 14): 3474-3484. DOI: 10.1242/jcs.103952

    [28]Barton V, Fisher N, Biagini GA, et al. InhibitingPlasmodiumcytochrome bc 1: a complex issue[J]. Curr Opin Chem Biol, 2010, 14(4): 440-446. DOI: 10.1016/j.cbpa.2010.05.005

    [29]Tucker EJ, Wanschers BF, Szklarczyk R, et al. Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression[J]. PLoS Genet, 2013, 9(12): e1004034. DOI: 10.1371/journal.pgen.1004034

    [30]Hildenbeutel M, Hegg EL, Stephan K, et al. Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation[J]. J Cell Biol, 2014, 205(4): 511-524. DOI: 10.1083/jcb.201401009

    [31]Mo YY. MicroRNA regulatory networks and human disease[J]. Cell Mol Life Sci, 2012, 69(21): 3529-3531. DOI: 10.1007/s00018-012-1123-1

    Received:2015-09-03;Revision accepted:2015-10-26

    Application of multi-locus sequence typing (MLST)in the identification ofEscherichiaalbertii

    LIU Xiang1,XU Yan-mei2,WANG Bin1,DENG Jian-ping1,XIAO Bo1,SUN Song-song1,ZHOU Yang1,XIONG Yan-wen2,WANG Hong1

    (1.ZigongCenterforDiseaseControlandPrevention,Zigong643000,China;2.StateKeyLaboratoryforInfectiousDiseasePreventionandControl,NationalInstituteforCommunicableDiseaseControlandPrevention,ChineseCenterforDiseaseControlandPrevention,Beijing102206,China)

    We studied the application of multilocus sequence typing (MLST) in the discovery and identification of a new pathogenEscherichiaalbertii. MLST was performed according to theE.coliMLST database using seven housekeeping genes. The 7 housekeeping genes of 30 suspectedEscherichiaalbertiistrains isolated from retail raw meats were amplified, sequenced and analysed by DNA star software. The nucleotide sequences were uploaded toE.coliMLST database to confirm the allele number and the sequence type (ST type). To analyse the phylogenetic relationships between the 30 strains and the reference strains ofE.coli,E.fergusonii,E.albertii,Shigellaspp. andSalmonellaentericaserovar Typhi, the phylogenetic tree was constructed with the neighbor-joining method of MEGA 6. Results showed that 30 strains were classified into 7 new STs (16 strains) and 4 known STs (14 strains), the analysis of N-J showed high homology withE.albertiistrain, while were highly divergent fromE.coli,Shigellasp.,E.fergusoniiandSalmonellaentericaserotype Typhi stains. MLST accurately determine the relationship between species ofE.albertiiat the level of housekeeping genes. And it is the first time of discovering and confirming the existence ofE.albertiiin China.

    multilocus sequence typing (MLST);E.albertii; new pathogens

    s: Wang Hong, Email: 460973389@qq.com; Xiong Yan-wen, Email: xiongyanwen@icdc.cn

    Effects of miR-36f on larval infection and development ofAscarissuum

    FENG Sheng-yong1,FU Jing-hua1,2,SHAO Chang-chun1,3,ZHU Xing-quan1,XU Min-jun1,2

    (1.StateKeyLaboratoryofVeterinaryEtiologicalBiology,KeyLaboratoryofVeterinaryParasitologyofGansuProvince,LanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciences,Lanzhou730046,China;2.CollegeofAnimalScience,SouthChinaAgriculturalUniversity,Guangzhou510642,China;3.CollegeofVeterinaryMedicine,YangzhouUniversity,Yangzhou225009,China)

    自貢市重點科技計劃項目(No.2013S06)和四川省衛(wèi)生和計劃生育委員會科研課題(No.150259)

    王 紅,Email: 460973389@qq.com; 熊衍文,Email: xiongyanwen@icdc.cn

    1.四川省自貢市疾病預(yù)防控制中心,自貢 643000; 2.中國疾病預(yù)防控制中心傳染病預(yù)防控制所,傳染病預(yù)防控制國家重點實驗室,北京 102206

    10.3969/j.issn.1002-2694.2015.11.009

    R378

    A

    1002-2694(2015)11-1033-04

    2015-02-05;

    2015-07-26

    Supported by the Zigong Key Science and Technology Program (No. 2013S06) and Grant from the Health and Family Planning Commission of Sichuan Province (No. 150259)

    猜你喜歡
    艾伯特株菌埃希菌
    拉長時間的公平
    做人與處世(2022年5期)2022-05-26 23:08:30
    It Couldn’t Be Done/by Edgar Albert Guest這是不可能的
    卷柏素對唑類藥物體外抗念株菌的增效作用
    3株耐鹽細菌對萘、菲、惹烯和苯并[α]芘的降解性能
    Clustering of Virtual Network Function Instances Oriented to Compatibility in 5G Network
    艾伯特·赫希曼的政治經(jīng)濟學(xué)思想述評
    522例產(chǎn)ESBLs大腸埃希菌醫(yī)院感染的耐藥性和危險因素分析
    產(chǎn)β-內(nèi)酰胺酶大腸埃希菌的臨床分布及耐藥性分析
    尿液大腸埃希菌和肺炎克雷伯菌I類整合子分布及結(jié)構(gòu)研究
    珠海地區(qū)婦幼保健院大腸埃希菌產(chǎn)ESBLs的基因型研究
    国产一区二区 视频在线| 国产淫语在线视频| 青草久久国产| 高清黄色对白视频在线免费看| 免费看不卡的av| 成人午夜精彩视频在线观看| 亚洲,欧美,日韩| 亚洲av日韩精品久久久久久密 | 国产精品99久久99久久久不卡 | 免费高清在线观看视频在线观看| 国产熟女午夜一区二区三区| 亚洲国产av新网站| 成人三级做爰电影| 91老司机精品| 国产精品久久久久成人av| 国产精品国产三级国产专区5o| 嫩草影视91久久| 久久亚洲国产成人精品v| 在线看a的网站| 国产97色在线日韩免费| 精品国产国语对白av| 亚洲国产成人一精品久久久| 十八禁网站网址无遮挡| 成人国产麻豆网| 看免费av毛片| 亚洲美女搞黄在线观看| 精品国产乱码久久久久久男人| 国产精品秋霞免费鲁丝片| 啦啦啦视频在线资源免费观看| 国产精品99久久99久久久不卡 | 国产成人av激情在线播放| 韩国av在线不卡| 亚洲精华国产精华液的使用体验| 国产精品无大码| 久久午夜综合久久蜜桃| 亚洲精品国产区一区二| 涩涩av久久男人的天堂| 婷婷色av中文字幕| 久久人人爽av亚洲精品天堂| 波野结衣二区三区在线| 美女大奶头黄色视频| bbb黄色大片| 2018国产大陆天天弄谢| 天美传媒精品一区二区| 免费在线观看视频国产中文字幕亚洲 | 大香蕉久久网| 亚洲av欧美aⅴ国产| av福利片在线| 欧美人与性动交α欧美精品济南到| 婷婷色综合大香蕉| 99精品久久久久人妻精品| 99久久精品国产亚洲精品| 夫妻午夜视频| 免费观看性生交大片5| 不卡av一区二区三区| 久久人人爽人人片av| 亚洲图色成人| 亚洲人成网站在线观看播放| 只有这里有精品99| 国产成人a∨麻豆精品| 韩国精品一区二区三区| 你懂的网址亚洲精品在线观看| 最新在线观看一区二区三区 | 十八禁人妻一区二区| 丰满乱子伦码专区| 日韩 亚洲 欧美在线| 两个人免费观看高清视频| 日韩av不卡免费在线播放| 成年女人毛片免费观看观看9 | 亚洲精品中文字幕在线视频| 色94色欧美一区二区| 精品国产一区二区久久| 在现免费观看毛片| 亚洲第一青青草原| 午夜日本视频在线| 久久久久人妻精品一区果冻| 韩国高清视频一区二区三区| 色94色欧美一区二区| 色婷婷av一区二区三区视频| 曰老女人黄片| 亚洲,欧美,日韩| 99香蕉大伊视频| 亚洲色图综合在线观看| 无遮挡黄片免费观看| 肉色欧美久久久久久久蜜桃| av一本久久久久| 亚洲熟女毛片儿| 欧美黄色片欧美黄色片| 亚洲av成人精品一二三区| 中文字幕高清在线视频| 亚洲第一av免费看| 街头女战士在线观看网站| xxxhd国产人妻xxx| 熟妇人妻不卡中文字幕| 免费观看av网站的网址| 午夜激情久久久久久久| 激情视频va一区二区三区| 亚洲成av片中文字幕在线观看| 午夜av观看不卡| 日日爽夜夜爽网站| 国产精品久久久av美女十八| 免费高清在线观看视频在线观看| av卡一久久| 精品人妻一区二区三区麻豆| 亚洲第一区二区三区不卡| 少妇的丰满在线观看| 老司机在亚洲福利影院| 91精品三级在线观看| 国产熟女午夜一区二区三区| 午夜福利影视在线免费观看| 日韩精品免费视频一区二区三区| 亚洲一卡2卡3卡4卡5卡精品中文| 毛片一级片免费看久久久久| h视频一区二区三区| 视频在线观看一区二区三区| 国产av国产精品国产| 久久人人97超碰香蕉20202| 黑丝袜美女国产一区| 精品久久蜜臀av无| 丰满饥渴人妻一区二区三| 成年人免费黄色播放视频| 亚洲美女视频黄频| 777久久人妻少妇嫩草av网站| 考比视频在线观看| 久久精品久久久久久噜噜老黄| 天天躁日日躁夜夜躁夜夜| 亚洲欧美色中文字幕在线| 久久久久久久久久久久大奶| 久久青草综合色| 99久久人妻综合| 免费观看av网站的网址| 综合色丁香网| 日韩 欧美 亚洲 中文字幕| 天天躁夜夜躁狠狠久久av| 人人澡人人妻人| av线在线观看网站| 男女边摸边吃奶| 精品少妇久久久久久888优播| 狠狠精品人妻久久久久久综合| 色网站视频免费| 亚洲成国产人片在线观看| 2021少妇久久久久久久久久久| 91成人精品电影| 欧美精品高潮呻吟av久久| 91精品三级在线观看| 久久久久久久大尺度免费视频| 黑人猛操日本美女一级片| 国产精品偷伦视频观看了| 亚洲欧美精品自产自拍| 看免费成人av毛片| 午夜免费男女啪啪视频观看| 三上悠亚av全集在线观看| 欧美日韩国产mv在线观看视频| 久久综合国产亚洲精品| 亚洲国产精品成人久久小说| 国产又色又爽无遮挡免| 成人黄色视频免费在线看| 国产免费一区二区三区四区乱码| 国产成人午夜福利电影在线观看| 久久这里只有精品19| 国产老妇伦熟女老妇高清| 宅男免费午夜| 国产精品亚洲av一区麻豆 | 十八禁人妻一区二区| 少妇猛男粗大的猛烈进出视频| 日本91视频免费播放| 麻豆av在线久日| 啦啦啦中文免费视频观看日本| 日本91视频免费播放| 亚洲精品视频女| 久久国产亚洲av麻豆专区| 欧美精品高潮呻吟av久久| 汤姆久久久久久久影院中文字幕| 免费观看性生交大片5| 在线观看人妻少妇| 免费观看a级毛片全部| 亚洲精品国产av成人精品| 成人午夜精彩视频在线观看| 久久久精品国产亚洲av高清涩受| 久久久久精品性色| videosex国产| 国产精品三级大全| 亚洲av在线观看美女高潮| 高清黄色对白视频在线免费看| 男女高潮啪啪啪动态图| av电影中文网址| 在线观看www视频免费| 精品久久久精品久久久| 亚洲欧美激情在线| 国产成人a∨麻豆精品| 一级毛片电影观看| 久久精品久久久久久噜噜老黄| 国产精品一国产av| 国产精品偷伦视频观看了| 日韩一区二区视频免费看| 熟女av电影| 青春草视频在线免费观看| 国产亚洲av片在线观看秒播厂| 免费黄网站久久成人精品| 亚洲视频免费观看视频| 男女边摸边吃奶| 久久精品久久精品一区二区三区| 美女午夜性视频免费| 国产一区二区三区av在线| 欧美人与性动交α欧美软件| 亚洲精品美女久久久久99蜜臀 | 亚洲欧美清纯卡通| 女性生殖器流出的白浆| 久久人人爽av亚洲精品天堂| h视频一区二区三区| 少妇被粗大猛烈的视频| 97精品久久久久久久久久精品| 久久女婷五月综合色啪小说| 国产精品女同一区二区软件| 男人添女人高潮全过程视频| 99香蕉大伊视频| 亚洲国产精品成人久久小说| 丝袜美腿诱惑在线| 最近中文字幕高清免费大全6| 国产一区有黄有色的免费视频| 两性夫妻黄色片| 欧美 亚洲 国产 日韩一| 亚洲久久久国产精品| 一本色道久久久久久精品综合| 黄网站色视频无遮挡免费观看| 国产男女超爽视频在线观看| 我的亚洲天堂| 欧美变态另类bdsm刘玥| 精品一区二区免费观看| 久久久精品区二区三区| 夫妻性生交免费视频一级片| 婷婷色综合大香蕉| 美女扒开内裤让男人捅视频| 又大又黄又爽视频免费| √禁漫天堂资源中文www| 国产成人免费观看mmmm| 一区在线观看完整版| 男女边摸边吃奶| 十八禁人妻一区二区| 国产极品天堂在线| 亚洲精品日韩在线中文字幕| 日韩av不卡免费在线播放| av片东京热男人的天堂| 国产亚洲最大av| 不卡av一区二区三区| 日韩 欧美 亚洲 中文字幕| av网站免费在线观看视频| 2018国产大陆天天弄谢| 黄色视频在线播放观看不卡| 国产精品久久久人人做人人爽| 超色免费av| av国产精品久久久久影院| av线在线观看网站| 精品免费久久久久久久清纯 | 婷婷色综合大香蕉| 热re99久久精品国产66热6| 亚洲国产精品一区二区三区在线| 99久久综合免费| 国产成人一区二区在线| 欧美中文综合在线视频| 久久国产精品男人的天堂亚洲| 久久精品久久久久久噜噜老黄| 侵犯人妻中文字幕一二三四区| 欧美激情高清一区二区三区 | 久久精品国产亚洲av高清一级| 丁香六月欧美| 亚洲精品美女久久av网站| 亚洲一级一片aⅴ在线观看| 不卡av一区二区三区| 男女午夜视频在线观看| 乱人伦中国视频| 国产欧美亚洲国产| 日韩不卡一区二区三区视频在线| 久久精品国产综合久久久| 免费日韩欧美在线观看| 日韩中文字幕欧美一区二区 | 亚洲国产欧美日韩在线播放| 欧美av亚洲av综合av国产av | 高清av免费在线| 日本猛色少妇xxxxx猛交久久| 精品第一国产精品| 久久 成人 亚洲| 老司机影院成人| 晚上一个人看的免费电影| 高清视频免费观看一区二区| 啦啦啦在线观看免费高清www| 99久久人妻综合| 男人操女人黄网站| 国产亚洲av高清不卡| 久久99一区二区三区| 亚洲国产最新在线播放| 老汉色av国产亚洲站长工具| 成人漫画全彩无遮挡| avwww免费| 亚洲免费av在线视频| 18禁观看日本| 最黄视频免费看| 韩国高清视频一区二区三区| 男女边摸边吃奶| 在现免费观看毛片| 国产成人精品久久久久久| 9191精品国产免费久久| 亚洲成色77777| 日韩成人av中文字幕在线观看| 久久久欧美国产精品| 欧美日韩亚洲国产一区二区在线观看 | 在线看a的网站| 亚洲国产精品一区三区| 国产在线一区二区三区精| 久久99一区二区三区| 亚洲国产欧美网| 精品少妇黑人巨大在线播放| 天堂8中文在线网| 精品一区二区三卡| 又大又爽又粗| 熟女少妇亚洲综合色aaa.| 成年人午夜在线观看视频| 少妇 在线观看| 欧美激情 高清一区二区三区| 性高湖久久久久久久久免费观看| 国产免费现黄频在线看| 国产成人精品久久久久久| 99香蕉大伊视频| 久久久精品94久久精品| 亚洲婷婷狠狠爱综合网| 国产 精品1| 国产成人啪精品午夜网站| 精品少妇久久久久久888优播| 亚洲成人一二三区av| 国产精品.久久久| 午夜久久久在线观看| 黄色 视频免费看| 一区二区三区精品91| 欧美精品亚洲一区二区| 亚洲一区二区三区欧美精品| 狂野欧美激情性bbbbbb| 香蕉丝袜av| av国产久精品久网站免费入址| 看十八女毛片水多多多| 好男人视频免费观看在线| 美女高潮到喷水免费观看| 最近中文字幕高清免费大全6| 欧美黑人欧美精品刺激| 精品一区二区三区av网在线观看 | 亚洲成国产人片在线观看| 十八禁高潮呻吟视频| 亚洲美女视频黄频| 国产爽快片一区二区三区| 亚洲一卡2卡3卡4卡5卡精品中文| 18禁观看日本| 国产麻豆69| 九九爱精品视频在线观看| 在线观看免费午夜福利视频| 国产深夜福利视频在线观看| 成人国产麻豆网| 18禁观看日本| 日韩一卡2卡3卡4卡2021年| 自线自在国产av| 少妇人妻 视频| 女人被躁到高潮嗷嗷叫费观| 在线观看免费午夜福利视频| av线在线观看网站| 国产熟女午夜一区二区三区| 美女国产高潮福利片在线看| 亚洲第一av免费看| 国产精品 国内视频| 最新的欧美精品一区二区| av网站在线播放免费| 好男人视频免费观看在线| 亚洲专区中文字幕在线 | 亚洲国产中文字幕在线视频| 亚洲精品久久午夜乱码| 国产精品国产三级国产专区5o| 欧美黑人精品巨大| 亚洲av日韩在线播放| 女人精品久久久久毛片| 91精品国产国语对白视频| 精品亚洲成国产av| 精品国产露脸久久av麻豆| 国产视频首页在线观看| 日韩精品免费视频一区二区三区| www.熟女人妻精品国产| 高清视频免费观看一区二区| 黄色怎么调成土黄色| 亚洲久久久国产精品| 熟女少妇亚洲综合色aaa.| 狠狠精品人妻久久久久久综合| 国产熟女午夜一区二区三区| 丰满饥渴人妻一区二区三| 天堂中文最新版在线下载| 精品少妇内射三级| 最近中文字幕2019免费版| 婷婷色综合www| 在线看a的网站| a 毛片基地| 亚洲一区二区三区欧美精品| 亚洲精品中文字幕在线视频| 午夜老司机福利片| 国产精品麻豆人妻色哟哟久久| 国产精品人妻久久久影院| 成年动漫av网址| www.熟女人妻精品国产| 中文字幕人妻丝袜一区二区 | 一级毛片黄色毛片免费观看视频| 五月天丁香电影| 老鸭窝网址在线观看| 亚洲五月色婷婷综合| 国产黄频视频在线观看| 晚上一个人看的免费电影| 视频区图区小说| 自线自在国产av| 色吧在线观看| 秋霞伦理黄片| 欧美精品av麻豆av| 久久鲁丝午夜福利片| 中文字幕高清在线视频| 免费女性裸体啪啪无遮挡网站| 国产老妇伦熟女老妇高清| 久久天躁狠狠躁夜夜2o2o | 国产精品二区激情视频| 日日摸夜夜添夜夜爱| 十八禁网站网址无遮挡| 欧美少妇被猛烈插入视频| 超碰97精品在线观看| 伦理电影大哥的女人| 免费观看a级毛片全部| 国产99久久九九免费精品| 婷婷成人精品国产| 国产精品二区激情视频| 哪个播放器可以免费观看大片| 中文字幕av电影在线播放| 国产深夜福利视频在线观看| 久久精品人人爽人人爽视色| 中文字幕人妻熟女乱码| 天堂8中文在线网| 国产成人欧美| 宅男免费午夜| 亚洲精品视频女| 欧美少妇被猛烈插入视频| 日本91视频免费播放| 久久精品久久久久久噜噜老黄| 亚洲精品成人av观看孕妇| av.在线天堂| 亚洲成人手机| 国产探花极品一区二区| 国产视频首页在线观看| 久久婷婷青草| 色吧在线观看| 老熟女久久久| 欧美人与性动交α欧美精品济南到| 秋霞在线观看毛片| 欧美精品一区二区大全| 亚洲综合精品二区| 老司机影院毛片| xxxhd国产人妻xxx| 久久亚洲国产成人精品v| 日本猛色少妇xxxxx猛交久久| 国产成人系列免费观看| 一本—道久久a久久精品蜜桃钙片| 伊人久久大香线蕉亚洲五| 女性被躁到高潮视频| 中文字幕精品免费在线观看视频| 美女高潮到喷水免费观看| 在线观看免费高清a一片| 久久毛片免费看一区二区三区| 亚洲国产欧美一区二区综合| 中文乱码字字幕精品一区二区三区| 久久人妻熟女aⅴ| 国产成人a∨麻豆精品| kizo精华| 搡老乐熟女国产| 日韩电影二区| 亚洲欧洲日产国产| 97在线人人人人妻| 亚洲国产av新网站| 久久人人97超碰香蕉20202| 日本av免费视频播放| 久久精品久久久久久久性| 只有这里有精品99| 一边摸一边做爽爽视频免费| 18在线观看网站| 美女国产高潮福利片在线看| 大香蕉久久网| 中文字幕最新亚洲高清| 欧美在线黄色| 久久久久精品人妻al黑| 777米奇影视久久| 乱人伦中国视频| 精品福利永久在线观看| 亚洲自偷自拍图片 自拍| 黄色视频不卡| www.熟女人妻精品国产| 一区二区日韩欧美中文字幕| 国产男女内射视频| 国产成人系列免费观看| 国产一区二区在线观看av| 999精品在线视频| 丝袜人妻中文字幕| 男人爽女人下面视频在线观看| 在线看a的网站| av国产久精品久网站免费入址| 另类亚洲欧美激情| 成人国产av品久久久| 999精品在线视频| 国产精品一国产av| 久久久精品区二区三区| 亚洲精品aⅴ在线观看| 九色亚洲精品在线播放| 日韩一本色道免费dvd| av电影中文网址| 男女之事视频高清在线观看 | 综合色丁香网| 在线精品无人区一区二区三| 国产精品女同一区二区软件| 中文字幕人妻丝袜一区二区 | 国产成人一区二区在线| 男女高潮啪啪啪动态图| 午夜福利乱码中文字幕| 最新的欧美精品一区二区| 国产精品一二三区在线看| 国产精品一国产av| 欧美成人精品欧美一级黄| av在线观看视频网站免费| 免费看av在线观看网站| 日本色播在线视频| 免费黄网站久久成人精品| 少妇人妻久久综合中文| 精品亚洲乱码少妇综合久久| 亚洲精品乱久久久久久| 国产精品香港三级国产av潘金莲 | 成人影院久久| 18禁国产床啪视频网站| 亚洲国产中文字幕在线视频| 国产一卡二卡三卡精品 | 亚洲图色成人| 日韩一卡2卡3卡4卡2021年| 在线观看一区二区三区激情| 精品国产一区二区久久| 伊人久久国产一区二区| 只有这里有精品99| 另类精品久久| 久久99精品国语久久久| 亚洲精品日本国产第一区| 成人毛片60女人毛片免费| 国产老妇伦熟女老妇高清| 视频在线观看一区二区三区| 国产亚洲av高清不卡| 日本色播在线视频| 国产日韩欧美亚洲二区| 国产男人的电影天堂91| 欧美变态另类bdsm刘玥| 搡老乐熟女国产| 欧美黄色片欧美黄色片| 国产成人av激情在线播放| 十八禁网站网址无遮挡| av卡一久久| 人体艺术视频欧美日本| 一本久久精品| 亚洲欧美一区二区三区黑人| 日本欧美国产在线视频| 久久久亚洲精品成人影院| 在线免费观看不下载黄p国产| 亚洲人成电影观看| 另类精品久久| av福利片在线| 18禁动态无遮挡网站| 精品国产一区二区久久| 精品亚洲成a人片在线观看| 国产成人91sexporn| 九九爱精品视频在线观看| av片东京热男人的天堂| 在线观看一区二区三区激情| 日韩av免费高清视频| 久久久欧美国产精品| 欧美日韩视频高清一区二区三区二| 久久久久精品人妻al黑| 一二三四中文在线观看免费高清| 女性生殖器流出的白浆| 丰满少妇做爰视频| 在线看a的网站| 国产老妇伦熟女老妇高清| 男女边摸边吃奶| 丝袜在线中文字幕| 国产97色在线日韩免费| 国产探花极品一区二区| 97人妻天天添夜夜摸| 日韩制服骚丝袜av| 亚洲,欧美,日韩| 丝袜脚勾引网站| 99热网站在线观看| 久久久久精品人妻al黑| 亚洲欧美精品综合一区二区三区| 极品人妻少妇av视频| 欧美人与善性xxx| 国产老妇伦熟女老妇高清| 亚洲国产av影院在线观看| 一本一本久久a久久精品综合妖精| 国产精品熟女久久久久浪| 人人妻人人澡人人看| 久久av网站| 老司机在亚洲福利影院| 亚洲av男天堂| 看免费成人av毛片| 久久人人爽人人片av| 国产男人的电影天堂91| 亚洲成色77777| 婷婷色av中文字幕| 免费av中文字幕在线| 狠狠婷婷综合久久久久久88av| 纯流量卡能插随身wifi吗| 久久午夜综合久久蜜桃| 永久免费av网站大全| 国产欧美日韩一区二区三区在线| 制服人妻中文乱码| 国产成人精品久久二区二区91 |