首页|H9N2亚型禽流感病毒HA-DCpep融合基因重组乳杆菌的构建及免疫原性分析

H9N2亚型禽流感病毒HA-DCpep融合基因重组乳杆菌的构建及免疫原性分析

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为了构建HA-DCpep融合基因重组乳杆菌并检测其表达产物的免疫原性,以pSIP409 HA质粒为模板,采用PCR方法将HA基因和DCpep基因融合.将目的基因克隆至pGEM-T载体中,进行测序.再将阳性基因亚克隆至大肠杆菌-乳杆菌穿梭表达载体pSIP409中,电转化入植物乳杆菌NC8中,经SppIP诱导后对表达产物进行Western-blot分析.结果显示,重组菌NC8-pSIP409-HA-DCpep的裂解物中出现大小约为64 ku的反应条带,大小与预期结果相符,表明重组融合蛋白具有良好的反应原性.重组菌NC8-pSIP409-HA-DCpep口服免疫SPF雏鸡后,在免疫后第14天可被检出特异性HI抗体,28d后达到高峰.结果表明,应用重组菌NC8-pSIP409-HA-DCpep免疫雏鸡能刺激机体产生特异性体液免疫应答反应,这为研制预防禽流感的重组乳杆菌口服制剂奠定了基础.
Construction and immunogenicity analysis of recombinant Lactobacillus plantarum NC8 expressing HA and DCpep genes of H9N2 subtype avian influenza virus
To construct the recombinant Lactobacillus plantarum NC8 for expression of HA-DCpep fusion gene and to detect the immunogenicity of fusion protein,the HA-DCpep gene was amplified by PCR and cloned into pGEM-T vector.The target gene was cloned into Escherichia coli-Lactobacillus shuttle expression vector pSIP409 after sequencing.The recombinant plasmid was electroporated into L.plantarum NC8,and was induced with SppIP.HA-DCpep expression was analyzed by Western-blot.About 64 ku ladder was detected from the lysates of recombinant NC8-pSIP409-HA-DCpep in Western-blot,indicating that the expressed fusion protein had strong reactinogenicity.SPF chicks were immunized orally with the recombinant NC8-pSIP409-HA-DCpep.HI antibody titers could be detected on day 14 post-injection and reached the peak after 28 days.In conclusion,the successfully-constructed NC8-pSIP409-HA-DCpep is able to induce specific humoral immune response in chicks.The recombinant NC8-pSIP409-HA-DCpep laid the foundation for the development of recombinant lactic acid bacteria oral preparations against avian influenza.

avian influenza virusHA-DCpep geneLactobacillus expression vectorimmunogenicity

石少华、杨文涛、叶丽萍、黄海斌、蔡若鹏、李雨宸、闫晓月、王春凤、杨桂连

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吉林农业大学 动物科学技术学院吉林省动物微生态制剂工程研究中心,吉林长春130118

吉林农业大学食药用菌教育部工程研究中心,吉林长春130118

禽流感病毒 HA-DCpep基因 乳杆菌表达载体 免疫原性

国家高技术研究发展计划(863)项目国家自然科学基金教育部新世纪优秀人才支持计划项目吉林省科技发展计划项目吉林省世行贷款农产品质量安全项目

2013AA102806,2011AA10A21531272541,31272552,81170358NCET-10-017520111816,200801042011-Y07

2014

中国兽医科学
中国农业科学院兰州兽医研究所

中国兽医科学

CSTPCDCSCD北大核心
影响因子：0.524
ISSN：1673-4696
年,卷(期)：2014.44(2)
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