抗肠道病毒71型外壳蛋白1卵黄抗体的制备和鉴定

The generation and identification of egg yolk antibody （IgY） against capsid protein VP1 of human enterovirus type 71 （EV71）

目的 克隆肠道病毒71型（EV71）BrCr株外壳蛋白1（VP1）基因,构建原核表达载体PET28a/VP1,表达VP1重组蛋白,以VP1免疫产蛋母鸡,获得抗VP1的鸡免疫球蛋白（IgY）.方法 培养Vero细胞,利用Vero细胞增殖肠道病毒EV71 BrCr株,提取病毒总RNA,利用RT-PCR扩增出VP1目的基因,目的基因插入克隆载体pGEM-T,提取重组质粒,经BamHⅠ、EcoRⅠ双酶切获得目的基因,插入原核表达载体pET-28a,重组子同时BamHⅠ,EcoRⅠ双酶切和测序鉴定,转化入感受态工程菌E.coli BL21（DE3）,获得阳性重组工程菌,经IPTG诱导获得表达融合蛋白,试剂盒纯化蛋白,经SDS-PAGE和Western blotting鉴定后,用VP1蛋白免疫开产母鸡,收集鸡蛋,利用EGG stractTM IgY Purification System试剂盒提取卵黄抗体IgY,间接ELISA测滴度,SDS-PAGE和Western blotting验证其表达量和免疫活性.结果 本实验成功扩增出肠道病毒EV71 BrCr株VP1基因,获得了含重组表达质粒pET28a/VP1的阳性工程菌株,经IPTG诱导能高效表达VP1蛋白,VP1经SDS-PAGE和Western blotting鉴定后,利用纯化后VP1蛋白免疫产蛋母鸡,提取的IgY经ELISA 鉴定后其滴度为1：104,SDS-PAGE和Western blotting证实为抗VP1的特异性IgY.结论 成功的从EV71 BrCr株扩增出VP1基因,构建了PET28a/VP1表达载体,获得高效表达融合蛋白,免疫产蛋母鸡后制备高效价抗VP1蛋白的特异性IgY.

Objective In order to clone the capsid protein VP1 gene of human enterovirus 71 （ EV71 ）, prokaryotic expression vector PET28a/VP1 was constructed to express VP1 recombinant protein, which was employed to immunize egglaying hens to obtain chicken immunoglobulin Y （IgY）. Methods Vero cells were cultured to proliferate the BrCr strains of EV71 in the cells and then the total RNA were extracted. A gene fragment encoding VP1 was obtained by RT -PCR amplification from EVT1 RNA and the target gene was inserted into cloning vector PGEM -T to extract the recombinant plasmids. The recombinants were double digested by BamHI and EcoRI to obtain gene of interest, which was inserted into prokaryotic expression vector PET28a and then the recombinants underwent simultaneous double digestion by BamHI and EcoRI as well to sequencing identification, and were transformed into the competent engineering bacteria E. coli BI21 （ DE3 ） to obtain positive recombinant engineering bacteria. Expression of fusion protein was induced by IPTG and the protein was purified by kit. Following the VPl identification by SDS - PAGE and Western blotting assays, puri- fied VP1 protein was used to immunize the egg -laying hens and eggs were collected. The IgY antibodies were extracted from these eggs using EGGstract？ IgY Purification System kit. The titre was determined by mediate ELISA and expression level and immunity activity were verified by SDS - PAGE and Western blotting. Results The capsid protein VP1 gene of human EV71 was successfully amplified and positive engineering strains containing recombinant expression plasmid pET28a/VP1 were obtained. Following IPTG induction, these strains could express VP1 fusion protein with high efficiency. Identification of VP1 by SDS - PAGE and Western blotting revealed that the immunized hens generated IgY antibodies against VP1 of human EV71. The egg yolk antibody obtained through immunized hens with VP1 showed a high titre of 1 ： 104. SDS - PAGE and Western blotting confirmed the specific IgY against VP1. Conclusion VP1 gene was successful- ly amplified from BrCr of EV71 and expression vector of PET28a/VPI was constructed, highly efficient fusion protein was obtained and high - efficiency specific IgY aginst VP1 protein was generated following immunization of egg - laying hens, which may provide a foundation for further studies on hand - foot - mouth disease.