猪源马链球菌兽疫亚种类M蛋白原核表达及其小鼠免疫保护力分析

周俊明，何孔旺，倪艳秀，祝昊丹，俞正玉，茅爱华，吕立新，温立斌，张雪寒，王小敏，汪伟，李彬，郭容利

(江苏省农业科学院兽医研究所，农业部兽用生物制品工程技术重点实验室，国家兽用生物制品工程技术研究中心，江苏南京210014)

摘要：为了原核表达猪源马链球菌兽疫亚种去除信号肽的类M蛋白质，并分析该蛋白质对小鼠的免疫保护力，克隆了马链球菌兽疫亚种CY菌株去除信号肽的类M蛋白质基因Szp，并将Szp基因插入pET28a表达载体，IPTG诱导，获得重组类M蛋白质，弗氏佐剂乳化重组类M蛋白质后，3次免疫小鼠，用同源菌株CY攻击。诱导获得分子量约5.8×10`4的重组蛋白质，免疫印迹结果表明该重组类M蛋白质能够被马链球菌兽疫亚种猪多抗识别。类M蛋白质免疫小鼠的存活率为60%，弗氏佐剂乳化灭活CY免疫小鼠的存活率为70%，PBS对照组小鼠攻毒后6 d内全部死亡。表达的类M蛋白质可以给予ICR小鼠部分的免疫保护力，保护效果略低于CY全菌灭活苗，提示可以对马链球菌兽疫亚种多种免疫保护性抗原进行联合，从而进一步提高对动物的免疫保护力。

关键词：马链球菌兽疫亚种；类M蛋白质；原核表达；疫苗

doi:10.3969/j.issn.1000-4440.2015.03.020

收稿日期：2014-10-08

基金项目：公益性行业(农业)科研专项经费项目(201303041)；江苏省自主创新探索性研究项目

作者简介：周俊明(1983-)，男，江苏东台人，硕士，助理研究员，主要研究方向为动物传染病防治。(Tel)025-84390988;(E-mail)zhoujm075@163.com

中图分类号：S858.282.61`+1

文献标识码：A

文章编号：1000-4440(2015)03-0590-05

Abstract：An open reading frame encoding M-like protein from strain CY of Streptococcus equi subsp. zooepidemicus of porcine origin without signal sequence was amplified by PCR using chromosomal DNA of CY as template. Amplicon was inserted into plasmid pET28a and transformed into Escherichia coli BL21 (DE3). A His-tag fusion protein SzP was then induced with IPTG. Mice were immunized three times with SzP and challenged by the homologous strain CY. The fusion protein SzP with molecular weight about 5.8×10`4 showed the specific immunoreactivity with pig antiserum against strain CY. Six SzP-vaccinated mice survied out of 10 tested mice, and the figure was 7 for whole cell bacterin-vaccinated mice. Meanwhile, the control mice vaccinated with PBS all died 6 days post-infection. It is indicated that the fusion protein SzP delivered certain protection against Streptococcus equi subsp. zooepidemicus.

Prokaryotic expression of M-like protein fromStreptococcusequisubsp.zooepidemicusof porcine origin and its immunoprotection in mice

ZHOU Jun-ming,HE Kong-wang,NI Yan-xiu,ZHU Hao-dan,YU Zheng-yu,MAO Ai-hua,LÜ Li-xin,WEN Li-bin,ZHANG Xue-han,WANG Xiao-min,WANG Wei,LI Bin,GUO Rong-li

(InstituteofVeterinaryMedicine,JiangsuAcademyofAgriculturalSciences/KeyLaboratoryofVeterinaryBiologicalEngineeringandTechnology,MinistryofAgriculture/NationalCenterforEngineeringResearchofVeterinaryBio-products,Nanjing210014,China)

Key words：Streptococcusequisubsp.zooepidemicus;M-like protein;prokaryotic expression;vaccine

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