猪肺炎支原体Mhp367蛋白不同区段与恢复期血清反应能力的比较

Comparison of the reactivity of convalescent sera to different regions of Mycoplasma hyopneumoniae Mhp367 protein

【背景】课题组前期研究发现猪肺炎支原体Mhp367蛋白是体液免疫显性蛋白,但该蛋白不同区段与猪肺炎支原体恢复期血清的反应能力尚不明确。【目的】鉴定Mhp367蛋白不同区段与猪肺炎支原体恢复期血清的反应能力。【方法】利用不同的引物组合扩增mhp367基因片段,扩增的片段连接pGEX-6P-1、pGEX-4T-3或pGEX-5X-3载体,转化大肠杆菌DH5α感受态细胞。提取的质粒经Bam H I和Xho I双酶切及测序确定重组质粒是否构建成功。正确的重组质粒转化大肠杆菌BL21(DE3)感受态细胞构建重组菌。重组菌经IPTG诱导和超声破菌后,经与谷胱甘肽beads结合和SDS-PAGE电泳检测目的蛋白表达情况。表达目的蛋白的重组菌破菌后上清包被谷胱甘肽板,ELISA方法鉴定Mhp367蛋白不同区段与猪肺炎支原体恢复期血清的反应能力。【结果】构建了9个能以可溶形式表达目的蛋白的重组菌;9个Mhp367蛋白片段均为体液免疫显性,第394-524位氨基酸区段与猪肺炎支原体恢复期血清反应最强,是一个良好的疫苗候选抗原区段。【结论】本研究为猪肺炎支原体基因工程亚单位疫苗的研发提供了候选抗原靶标。

[Background]Previous studies in our laboratory have demonstrated that Mycoplasma hyopneumoniae Mhp367 protein is a humoral immunodominant proteinic antigen.However,the reactivity of different regions of Mhp367 to the convalescent sera remains unclear.[Objective]The purpose of this study is to identify the reactivity of different regions of Mhp367 to the convalescent sera.[Methods]Nine DNA fragments of mhp367 were amplified with different primer combinations.The amplified products were respectively ligated into pGEX-6P-1,pGEX-4T-3,or pGEX-5X-3,and the ligation products were transformed into Escherichia coli DH5αcompetent cells.The constructed plasmids were extracted from the cells and confirmed by digestion with Bam HⅠand XhoⅠand nucleotide sequencing.The identified plasmids were then transformed into E.coli BL21(DE3)to express target proteins(different fragments of Mhp367)under the induction of IPTG.After ultrasonic disruption of the recombinant bacteria,the protein was purified with glutathione-conjugated agarose beads.The expression of proteins was visualized by SDS-PAGE.ELISA was employed to identify the reactivity between different fragments of Mhp367protein and convalescent sera.[Results]Nine recombinant strains that could express different fragments of Mhp367 in soluble form were successfully constructed.All of the nine fragments were humoral immunodominant.The region aa 394–524 in Mhp367 had the strongest reactivity with convalescent sera and was a good antigen fragment for vaccine candidates.[Conclusion]This study provides a candidate of genetic engineering subunit vaccines against M.hyopneumoniae.