牛乳头瘤病毒2型L1基因的原核表达及多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2019.06.029

摘要/Abstract

摘要：

试验旨在表达牛乳头瘤病毒（BPV）的L1衣壳蛋白，并制备其多克隆抗体。对疑似牛乳头状瘤的病料进行病理组织学检查，采用L1基因简并引物FAP59/FAP64进行扩增，然后设计特异性引物，将衣壳蛋白L1基因亚克隆至pET-30a （+）表达载体中，构建重组原核表达载体pET30a-BPV2-L1，在E.coli Rosetta^TM（DE3） pLysS宿主菌中表达重组蛋白rBPV2-L1，并以纯化的rBPV2-L1蛋白为免疫原制备兔抗BPV2-L1蛋白多克隆抗体，随后间接ELISA检测其效价，并进行间接免疫荧光试验分析。病理组织切片观察结果显示，病变部分表皮细胞增生、角质过度并出现细胞挖空等BPV感染的组织病变情况；序列分析确定BPV分离株的基因型为2型，L1基因编码区长1 494 bp，可编码一个含有497个氨基酸的衣壳蛋白，蛋白分子质量为55.5 ku；与各型BPV的L1氨基酸序列系统进化树分析结果显示，其与BPV2-SW01（GenBank登录号：KC878306.1）、BPV2（GenBank登录号：KX113620.1）处于同一遗传进化分支，且属于Delta属成员；诱导表达的rBPV2-L1蛋白主要以包涵体形式存在于沉淀中；间接ELISA法检测多克隆抗体效价高达1：163 840，间接免疫荧光分析表明，兔抗BPV2-L1多克隆抗体能与瞬时表达的BPV2-L1蛋白发生特异性反应。以上结果证实，本研究成功克隆、表达了BPV2 L1基因，且重组蛋白rBPV2-L1具有较好的免疫原性，所制备的BPV2-L1蛋白多克隆抗体具有良好的免疫活性，为进一步研究BPV2亚单位疫苗奠定基础。

关键词: 牛乳头瘤病毒(BPV); L1基因; 原核表达; 多克隆抗体

Abstract:

The test was aimed to express the L1 capsid protein of bovine papillomavirus (BPV),and prepare its polyclonal antibodies.Histopathological examination was conducted using the cutaneous papilloma samples of cattle.The L1 gene degenerate primer FAP59/FAP64 was used for amplification,and then specific primers were designed.The capsid protein L1 gene was subcloned into pET-30a(+) expression vector to construct recombinant prokaryotic expression vector pET30a-BPV2-L1.The recombinant protein rBPV2-L1 was expressed in E.coli Rosetta^TM(DE3)pLysS host strain,and the rabbit anti-BPV2-L1 protein polyclonal antibody was prepared by using purified rBPV2-L1 protein as immunogen,and then the titer was detected by indirect ELISA,indirect immunofluorescence assay was performed.The pathological tissue section observation showed that the epidermal cells proliferated,the keratin was excessive,and the tissue lesions of BPV infection such as cell hollowing occurred.Sequence analysis confirmed that the genotype of the BPV isolated strain was type 2,and the coding region of L1 gene was 1 494 bp,which could encode a capsid protein containing 497 amino acids with a molecular mass of 55.5 ku.Phylogenetic tree analysis with L1 amino acid sequence of each type of BPV showed that it was in the same genetic evolution branch as BPV2-SW01 (GenBank accession No.:KC878306.1) and BPV2 (GenBank accession No.:KX113620.1),and belonged to the genus Delta.The induced expression of rBPV2-L1 protein was mainly present in the precipitate in the form of inclusion bodies.The indirect ELISA assay results showed that the polyclonal antibody titer was as high as 1:163 840.Indirect immunofluorescence analysis results showed that the rabbit anti-BPV2-L1 polyclonal antibody reacted specifically with the transiently expressed BPV2-L1 protein.The above results confirmed that the BPV2 L1 gene was successfully cloned and expressed in this study,and the recombinant protein rBPV2-L1 had good immunogenicity.The prepared polyclonal antibody against BPV2-L1 protein had good immunological activity,and laid the foundation for further study of the BPV2 subunit vaccine.

Key words: bovine papilloma virus(BPV); L1 gene; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺3

杜继文, 刘悦, 宋志峰, 鲁兆祥, 徐玮程, 李明珠, 高明春, 王君伟. 牛乳头瘤病毒2型L1基因的原核表达及多克隆抗体的制备[J]. 《中国畜牧兽医》, 2019, 46(6): 1808-1815.

DU Jiwen, LIU Yue, SONG Zhifeng, LU Zhaoxiang, XU Weicheng, LI Mingzhu, GAO Mingchun, WANG Junwei. Prokaryotic Expression of Bovine Papillomavirus Type 2 L1 Gene and Preparation of Polyclonal Antibody[J]. , 2019, 46(6): 1808-1815.

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