猪流行性腹泻病毒COE基因重组乳酸菌的构建及免疫原性分析
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- 英文论文题名：Construction and Immunogenicity Analysis of Recombinant Lactococcus lactis Expressing COE Protein of Porcine Epidemic Diarrhea Virus
- 论文作者：杨洁 ; 张以芳
- 英文论文作者：Yang Jie ; Zhang Yifang
- 年：2016
- 作者机构：云南农业大学动物科学技术学院;
- 论文关键词：PEDV ; COE基因 ; 乳酸乳球菌 ; NICE系统 ; 口服疫苗
- 英文论文关键词：PEDV ; COE gene ; Lactococcus lactis ; Nisin controlled expression system ; oral vaccine
- 会议召开时间：2016-11-04
- 会议录名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会论文集
- 语种：中文
- 分类号：S852.651
- 学会代码：ZGXJ
- 会议名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会
- 会议地点：中国山东青岛
- 主办单位：中国畜牧兽医学会动物微生态学分会
- 学会名称：中国畜牧兽医学会
- 页数：1
- 文件大小：107k
- 原文格式：O
- 会议级别：全国
摘要
本研究以乳酸乳球菌乳链菌肽表达(Nisin controlled expression,NICE)系统为抗原递呈载体,以PEDV COE基因片段为靶基因,构建了表达PEDV COE基因的重组乳酸乳球菌,并对其免疫原性进行了初步评价。根据GenBank中已发表的PEDV序列,设计1对引物,对云南某猪场采集的疑似患PED病猪肠病料进行RT-PCR扩增,获得778bp的PEDV部分保护性抗原基因,将其克隆至pMD19-T后测序,核苷酸序列同PEDV参考序列同源性为94.5%~99.6%。根据目的基因和表达载体特点,设计1对引物,以pMD19-S为模版扩增COE基因(约510bp)。将COE基因与乳酸乳球菌表达载体pNZ8148进行连接,电转化乳酸乳球菌NZ9000,得到重组菌pNZ8148-COE/NZ9000。重组菌用Nisin诱导后,SDS-PAGE可检测到分子量约20kDa的蛋白,与预期蛋白大小一致;间接免疫荧光试验表明,重组菌表达蛋白位于菌体表面且具有反应原性。将构建的重组乳酸乳球菌pNZ8148-COE/NZ9000口服免疫初生仔猪,加强免疫后10d采集免疫动物的血液、粪便样品,处理后用MTS法和ELISA法检测T淋巴细胞转化情况、特异性抗体(IgG和sIgA)及细胞因子(IL-4和IFN-γ),评价重组菌刺激产生的体液免疫和细胞免疫水平。同时,在免疫前后测定仔猪体重,评价其对仔猪生长发育的影响。试验结果经统计分析表明,各免疫组对仔猪体重增加没有负影响;含有PEDV COE基因的重组乳酸菌口服免疫仔猪后,淋巴细胞增殖试验刺激指数SI、血清抗体IgG、粪便中的sIgA、外周血淋巴细胞悬液中IL-4和IFN-γ含量均高于PBS对照组和空载体对照组,组间差异显著(P<0.05)。由此说明,重组菌pNZ8148-COE/NZ9000具有良好的免疫原性,并能诱导机体产生黏膜免疫反应和系统免疫反应。本研究为进一步开发新型、有效的PEDV口服疫苗奠定了基础。
In this study,we constructed a recombinant Lactococcus lactis NZ9000 expressing COE,which took the Nisin controlled expression(NICE) system as an antigen delivery vehicle,PEDV COE gene as the target gene,and evaluated immunogenicity of the recombinant strain.According to the complete sequence of PEDV published in GenBank,a pair of specific primers was designed and synthesized.RT-PCR was conducted on intestine and its content of piglets with diarrhea from a farm in Yunnan.The partial S glycoprotein gene of PEDV was cloned into pMD19-T and sequenced.The gene consisted of 778 bp and shared 94.5%~99.6%nucleotide homology with the PEDV reference sequence.We designed another pair of primers according with the target gene and considering the characters of expression vector plasmid.About510 bp gene fragment(COE) was amplified by PCR using the plasmid pMD19-S.The COE gene was subcloned into the Lactococcus lactis vector pNZ8148 and transformed into Lactococcus lactis NZ9000 by electroporation,generating pNZ8148-COE/NZ9000.The recombinant protein was detected by SDS-PAGE and IFA experiments after the bacteria induced by Nisin.The result indicated that the molecular weight of the expressed recombinant protein was about 20 kDa as expected,the protein maintain the antigenicity of PEDV,moreover the protein was secreted and located on the surface of the bacteria.The recombinant Lactococcus lactis named pNZ8148-COE/NZ9000 were cultured and induced by Nisin,then orallied immunization to newborn piglets.The lymphocyte proliferation stimulating index and specific antibody(IgG and sIgA),cytokine(IL-4 and IFN-γ) were measured by MTS and ELISA respectively,and the degree of humoral immunity and cellular immunity induced by the recombinant bacteria were evaluated.Simultaneously,the body weights of piglets in different groups were weighed to evaluate the influence on the growth of piglets immunized.The results showed that the recombinant Lactococcus lactis had no negative effect on the weight gain for the immunized groups of piglets.The lymphocyte proliferation stimulating index,IgG in the serum,slgA in the excrement,IL-4 and IFN-y of test piglets immunized with recombinant bacteria were all significant higher than that of the control groups.Significant differences were seen among the groups(P<0.05).All the results indicated that the recombinant strain has better immunogenicity and can elicit stronger specific mucosal immune responses and systematic immune responses.All theses work established a good foundation for futher study on the new and effective recombinant oral vaccine of PEDV.
In this study,we constructed a recombinant Lactococcus lactis NZ9000 expressing COE,which took the Nisin controlled expression(NICE) system as an antigen delivery vehicle,PEDV COE gene as the target gene,and evaluated immunogenicity of the recombinant strain.According to the complete sequence of PEDV published in GenBank,a pair of specific primers was designed and synthesized.RT-PCR was conducted on intestine and its content of piglets with diarrhea from a farm in Yunnan.The partial S glycoprotein gene of PEDV was cloned into pMD19-T and sequenced.The gene consisted of 778 bp and shared 94.5%~99.6%nucleotide homology with the PEDV reference sequence.We designed another pair of primers according with the target gene and considering the characters of expression vector plasmid.About510 bp gene fragment(COE) was amplified by PCR using the plasmid pMD19-S.The COE gene was subcloned into the Lactococcus lactis vector pNZ8148 and transformed into Lactococcus lactis NZ9000 by electroporation,generating pNZ8148-COE/NZ9000.The recombinant protein was detected by SDS-PAGE and IFA experiments after the bacteria induced by Nisin.The result indicated that the molecular weight of the expressed recombinant protein was about 20 kDa as expected,the protein maintain the antigenicity of PEDV,moreover the protein was secreted and located on the surface of the bacteria.The recombinant Lactococcus lactis named pNZ8148-COE/NZ9000 were cultured and induced by Nisin,then orallied immunization to newborn piglets.The lymphocyte proliferation stimulating index and specific antibody(IgG and sIgA),cytokine(IL-4 and IFN-γ) were measured by MTS and ELISA respectively,and the degree of humoral immunity and cellular immunity induced by the recombinant bacteria were evaluated.Simultaneously,the body weights of piglets in different groups were weighed to evaluate the influence on the growth of piglets immunized.The results showed that the recombinant Lactococcus lactis had no negative effect on the weight gain for the immunized groups of piglets.The lymphocyte proliferation stimulating index,IgG in the serum,slgA in the excrement,IL-4 and IFN-y of test piglets immunized with recombinant bacteria were all significant higher than that of the control groups.Significant differences were seen among the groups(P<0.05).All the results indicated that the recombinant strain has better immunogenicity and can elicit stronger specific mucosal immune responses and systematic immune responses.All theses work established a good foundation for futher study on the new and effective recombinant oral vaccine of PEDV.
引文