H6N6亚型禽流感病毒N6基因的原核表达与多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2018.11.002

摘要/Abstract

摘要：

为表达H6N6亚型禽流感病毒（avian influenza virus，AIV）神经氨酸酶（NA）蛋白，并制备多克隆抗体，本试验根据GenBank中AIV N6基因序列（登录号：MG434500）设计特异性引物，对贵州地区分离的H6N6亚型AIV贵州株进行N6基因PCR扩增，将其克隆到原核表达载体pET-32a （+）中，构建重组原核表达载体pET-32a-N6，转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导表达His-N6重组蛋白，将诱导产物超声破碎离心后，利用镍柱对重组蛋白进行纯化，并利用SDS-PAGE和Western blotting对表达蛋白进行双重鉴定，将纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体，并通过间接ELISA检测其效价。结果显示，AIV贵州株N6基因编码区长1 380 bp，可编码459个氨基酸，其中175－207 bp缺失33个核苷酸；重组质粒pET-32a-N6经双酶切鉴定分别获得大小为5 900 bp左右的载体条带和1 380 bp左右的目的基因条带，成功构建了pET-32a-N6重组质粒；蛋白超声破碎后经SDS-PAGE发现，重组蛋白主要存在于沉淀中，以包涵体形式存在，蛋白分子质量约为70 ku，与预期结果一致；纯化后的重组蛋白经SDS-PAGE和Western blotting双重鉴定，均在70 ku处出现条带，说明纯化的蛋白为重组蛋白pET-32a-N6，表达产物具有免疫学活性，包涵体经变性、复性处理，重组表达蛋白分别被His抗体和兔源抗N6多克隆抗体所识别。间接ELISA检测其效价高于1∶3 200。以上结果表明，试验成功克隆、表达了H6N6亚型AIV的N6基因，所制备的N6蛋白多克隆抗体具有良好的免疫活性，能被His抗体和兔源抗N6多克隆抗体所识别。

关键词: 禽流感病毒(AIV); N6亚型; 原核表达; 多克隆抗体

Abstract:

This experiment was aimed to express recombinant neuraminidase(NA) protein of H6N6 subtype avian influenza virus (AIV) isolated from Guizhou province,and prepared polyclonal antibody against NA protein of AIV.The specific primers were designed according to N6 gene of AIV in GenBank(accession No.:MG434500),and used for amplification of N6 gene by PCR.PCR product was cloned into the expression vector pET-32a(+).The soluble recombinant protein was expressed in E.coli BL21(DE3) with IPTG.The expression product was sonicated and purified by nickel column.The recombinant proteins were identified by SDS-PAGE and Western blotting.The polyclonal antibody was prepared from the rabbit immunized with purified recombinant protein.The titer of the antibody was detected by indirect ELISA.The results showed that coding region of N6 gene was 1 380 bp,which encoded 459 amino acids.There were 33 nucleotides deletion from position 175 to 207 bp at N6 gene stalk.The recombinant plasmid pET-32a-N6 was identified by double enzyme digestion,it appeared about 5 900 bp vector band and about 1 380 bp target band,pET-32a-N6 recombinant plasmid was successfully constructed;SDS-PAGE analysis result showed the molecular weight of the recombinant N6 protein was about 70 ku which consistented with expected result.It showed that the target gene was successfully expressed in the form of inclusion body.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and showed a band of 70 ku in size,indicating that the purified protein was the recombinant protein pET-32a-N6.The purified protein of denaturation and renaturation could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum,respectively.The titer of the antibody was about 1:3 200 by detection of indirect ELISA.All the results indicated that N6 gene of H6N6 subtype AIV was successfully cloned and expressed,the prepared anti-N6 showed good immune activity,and the protein purified could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum.

Key words: avian influenza virus (AIV); N6 subtype; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺7

万润, 华敏, 龙立书, 贺欣薇, 罗引幸, 周碧君, 王开功, 程振涛, 文明. H6N6亚型禽流感病毒N6基因的原核表达与多克隆抗体制备[J]. 《中国畜牧兽医》, 2018, 45(11): 2989-2995.

WAN Run, HUA Min, LONG Lishu, HE Xinwei, LUO Yinxing, ZHOU Bijun, WANG Kaigong, CHENG Zhentao, WEN Ming. Prokayotic Expression and Polyclonal Antibody Preparation of N6 Gene of H6N6 Subtype Avian Influenza Virus[J]. , 2018, 45(11): 2989-2995.

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