CAJ | 학술논문

为开展猪轮状病毒（PRV）及其VP4蛋白相关特性研究，参照GenBank所收录的猪轮状病毒JL94、OSU等株序列设计一对特异性引物，从实验室分离鉴定的猪轮状病毒DN30209株扩增约2330 bp VP4全长基因，并成功构建重组质粒VP4-pMD18-T。重组质粒经测序、序列比对及分析。结果表明，DN30209株VP4全基因长2331 bp，与参考株JL94的核苷酸同源性为99.7%，氨基酸同源性为99.5%。将VP4基因连接到pGEX-6P-1原核表达载体中，构建并筛选出阳性原核表达载体重组质粒VP4-pGEX-6P-1。阳性重组质粒转化Rosetta宿主菌感受态细胞中，经IPTG诱导，获得以包涵体形式表达的重组蛋白，融合蛋白大小约为112 ku，与预期大小相符。回收、纯化并复性该蛋白，将其作为免疫原免疫大白兔，制备兔抗PRV VP4全长蛋白多克隆抗体，间接ELISA测定多抗效价到1??105，间接免疫荧光（IFA）和免疫印迹（Western blot）检测表明该多抗均可与PRV具有很好的抗原抗体反应性，为猪轮状病毒研究提供有效实验材料。
위개전저륜상병독（PRV）급기VP4단백상관특성연구，삼조GenBank소수록적저륜상병독JL94、OSU등주서렬설계일대특이성인물，종실험실분리감정적저륜상병독DN30209주확증약2330 bp VP4전장기인，병성공구건중조질립VP4-pMD18-T。중조질립경측서、서렬비대급분석。결과표명，DN30209주VP4전기인장2331 bp，여삼고주JL94적핵감산동원성위99.7%，안기산동원성위99.5%。장VP4기인련접도pGEX-6P-1원핵표체재체중，구건병사선출양성원핵표체재체중조질립VP4-pGEX-6P-1。양성중조질립전화Rosetta숙주균감수태세포중，경IPTG유도，획득이포함체형식표체적중조단백，융합단백대소약위112 ku，여예기대소상부。회수、순화병복성해단백，장기작위면역원면역대백토，제비토항PRV VP4전장단백다극륭항체，간접ELISA측정다항효개도1??105，간접면역형광（IFA）화면역인적（Western blot）검측표명해다항균가여PRV구유흔호적항원항체반응성，위저륜상병독연구제공유효실험재료。
To study porcine rotavirus (PRV) VP4 protein levels related experiments,we refered to GenBank PRV JL94, OSU strains sequences, designed a pair of specific primers, the complete length of VP4 gene about 2 330 bp was cloned from the separation strain of DN30209 of our lab. Expanding product was constructed into a recombinant plasmid VP4-pMD18-T successfully. By sequencing, the sequence alignment and analysis, the results showed that the VP4 gene complete length of DN30209 was 2 331 bp, compared with JL94 strains, nucleotide homology was 99.7%, amino acid homology was 99.5%. The VP4 gene was connected with prokaryotic expression vector pGEX-6P-1, constructed and screened the positive prokaryotic expression vector recombinant plasmid, marked as VP4-pGEX-6P-1. Following, positive recombinant plasmid was transformed into Rosetta competent cell, by IPTG induction, we have got the expression of recombinant proteins in the form of inclusion body, the fusion protein size is about 113 ku, consistent with the expected size. The protein Retrieved and purified, was used as immunogen to the white rabbit, and then, rabbit anti-VP4 protein polyclonal antibody was preparated, and detected biological activity. The results showed that: the titer of polyclonal antibody was approximately 1??105, moreover, the polyclonal antibody, which has a good antigen-antibody reactivity with PRV, are available for IFA and WB, so, the PRV VP4 polyclonal antibody is an effective experimental material for porcine rotavirus research.