小反刍兽疫病毒H蛋白的原核表达及免疫原性测定

doi:10.19586/j.2095-2341.2021.0039

生物技术进展 ›› 2021, Vol. 11 ›› Issue (6): 770-776.DOI: 10.19586/j.2095-2341.2021.0039

小反刍兽疫病毒H蛋白的原核表达及免疫原性测定

李丹(), 宋浩志, 高维崧, 刘兴健, 张志芳, 李轶女()

中国农业科学院生物技术研究所，北京 100081

收稿日期:2021-03-25 接受日期:2021-06-02 出版日期:2021-11-25 发布日期:2021-11-26
通讯作者: 李轶女
作者简介:李丹 E-mail：lidancaas@163.com；
基金资助:
国家重点研发计划项目(2016YFD0500108);国家自然科学基金项目(32072796)

Prokaryotic Expression and Immunogenicity Detection of the H Protein of Peste des Petits Ruminants Virus

Dan LI(), Haozhi SONG, Weisong GAO, Xingjian LIU, Zhifang ZHANG, Yinyu LI()

Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2021-03-25 Accepted:2021-06-02 Online:2021-11-25 Published:2021-11-26
Contact: Yinyu LI

摘要/Abstract

摘要：

小反刍兽疫是由小反刍兽疫病毒（peste des petits ruminants virus，PPRV）引起的急性、接触性传染病，对我国的畜牧业发展造成了严重的影响，目前主要通过疫苗进行防控。为检测PPRV主要抗原蛋白血凝素（hemagglutinin，H）蛋白的免疫原性，从GenBank数据库中查找了近年来公布的H蛋白氨基酸序列，选择1条符合我国流行趋势的序列，对其基因序列进行优化合成后克隆至pET28a载体上。筛选出表达量高的Rosetta（DE3）菌株进行表达，表达产物经SDS?PAGE、Western Blot及质谱分析鉴定。经镍柱亲和层析纯化出单一H蛋白，取20 μg与佐剂混合后免疫小鼠，收集血清进行抗体效价检测，结果显示，血清中H蛋白抗体滴度在二免2周时达到1∶6 400，表明H蛋白具有较好的免疫原性。进一步对二免2周时血清进行中和抗体检测显示，小鼠血清对PPRV疫苗株具有中和效应，中和效价不超过1∶40。研究结果对H蛋白用于PPRV疫苗的研发提供了理论依据。

关键词: 小反刍兽疫病毒, 血凝素蛋白, 疫苗, 大肠杆菌表达系统

Abstract:

Peste des petits ruminants is an acute and contagious infectious disease caused by the peste des petitis ruminant virus （PPRV）， which has a serious impact on the development of China's animal husbandry. At present， vaccination is the main method of prevention and control. In order to detect the main antigen protein hemagglutinin （H） protein of PPRV， searched its amino acid sequence published in recent years from the GenBank databases， and selected a sequence that conformed to the popular trend in China. The gene sequence was optimized and synthesized， and then cloned into the pET28a vector. The high?expressing Rosetta （DE3） strain was selected for expression and the product was identified by SDS?PAGE， Western Blot and mass spectrometry analysis. A single H protein was purified by nickel column affinity chromatography， and 20 μg H protein was mixed with adjuvant to immunize mice， and serum was collected for antibody titer detection. The results showed that the serum antibody titer reached 1：6 400 after two weeks of the second immunization， indicating that H protein has good immunogenicity. Further testing of neutralizing antibodies on this serum showed that it had a neutralizing effect on the PPRV vaccine strain， and the neutralizing titer did not exceed 1：40. The results provide a theoretical basis for the use of H protein in the development of PPRV vaccines.

Key words: peste des petits ruminants virus, hemagglutinin protein, vaccine, Escherichia coli expression system

中图分类号:

S852

李丹, 宋浩志, 高维崧, 刘兴健, 张志芳, 李轶女. 小反刍兽疫病毒H蛋白的原核表达及免疫原性测定[J]. 生物技术进展, 2021, 11(6): 770-776.

Dan LI, Haozhi SONG, Weisong GAO, Xingjian LIU, Zhifang ZHANG, Yinyu LI. Prokaryotic Expression and Immunogenicity Detection of the H Protein of Peste des Petits Ruminants Virus[J]. Current Biotechnology, 2021, 11(6): 770-776.

图/表 6

图1 目的基因的PCR扩增及重组质粒的BamHⅠ、EcoRⅠ酶切鉴定A：目的基因的PCR扩增。PPRV H—以pUC57?PPRV H为模板的PCR扩增产物。B：重组质粒的BamH Ⅰ、EcoR Ⅰ酶切鉴定。pET 28a?PPRV H?1～2—重组质粒pET 28a?PPRV H经BamH Ⅰ、EcoRⅠ双酶切后产物。

Fig.1 PCR amplification of the target gene and BamH Ⅰ and EcoR Ⅰ digestion identification of the recombinant plasmid

图2 重组蛋白在Rosetta菌株中的诱导表达

Fig.2 Induced expression of recombinant protein in Rosetta strains

图3 重组蛋白的镍柱亲和纯化注：NTA100及NTA250为PPRV H蛋白的不同浓度咪唑洗脱样品。

Fig. 3 Nickel column affinity purification of recombinant protein

图4 重组蛋白的Western blot分析注：pET28a—空载体经表达纯化后所得样品；pET28a?PPRV H?1~3—pET28a?PPRV H经表达纯化后所得蛋白样品。

Fig.4 Western blot analysis of recombinant protein

图5 重组蛋白免疫小鼠后引起的抗体效价检测

Fig.5 Detection of antibody titer caused by recombinant protein immunization in mice

图6 不同稀释度血清抗体对PPRV病毒的中和效应

Fig.6 Neutralizing effect of different concentrations of serum antibodies on PPRV virus

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小反刍兽疫病毒H蛋白的原核表达及免疫原性测定

Prokaryotic Expression and Immunogenicity Detection of the H Protein of Peste des Petits Ruminants Virus

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