猪鼻支原体表面可变脂蛋白VlpB黏附宿主细胞功能研究

The function of the variable lipoprotein B of Mycoplasma hyorhinis in adherence to host cell

猪鼻支原体表面可变脂蛋白(Vlp)家族在黏附宿主细胞中起重要作用。为研究Vlp家族成员VlpB的黏附细胞功能,包括不同区段的黏附能力,以及其Ⅲ区重复片段的重复次数对黏附能力的影响。本研究利用间接免疫荧光和微孔板黏附试验验证了VlpB的黏附细胞能力。然后根据GenBank中登录的vlpB基因序列,构建了仅含Ⅱ区基因的重组质粒pET-32a(+)/vlpBn,诱导表达后纯化,获得纯度较高的重组蛋白Vlp B0;同时化学合成Ⅲ区重复片段PepVlpB并与KLH偶联;进一步合成Ⅲ区重复次数分别为3、6、9、12的一系列vlpB基因,构建pET-VlpB,诱导表达获得相应的重组蛋白VlpB3、VlpB6、VlpB9和VlpB12。利用微孔板黏附试验定量检测上述重组蛋白和偶联蛋白的细胞黏附能力。结果显示,仅含Ⅱ区的重组蛋白Vlp B0及含Ⅲ区重复片段的偶联蛋白KLH-PepVlpB可黏附宿主细胞;比较不同Ⅲ区重复次数的VlpB蛋白的黏附水平,结果显示,随着Ⅲ区重复次数增多,VlpB黏附能力逐渐下降。本研究证实Vlp B作为猪鼻支原体的黏附因子之一,其Ⅱ区和Ⅲ区均含有黏附位点,而整体分子的黏附能力随Ⅲ区重复次数增加而降低。本研究结果为猪鼻支原体黏附及致病机制研究提供启示。

The surface variable lipoprotein (vlp)family plays an important role in mediating cytoadhesion of Mycoplasma hyorhinis.Herein,we studied the cytoadhesive function of one of the vlp family member,vlpB,particularly the cytoadhesive ability of different vlpB regions and how the repeated times of repetitive unit in region Ⅲ impact on the function.Initially,the cytoadhesive fimction of vlpB was identified by indirect immuofluoresceence assay (IFA)and microtiter plate adherence assay. Then,a recombinant plasmid pET-vlpB0containing the DNA sequence encoding region Ⅱ of vlpB was constructed according to the sequence available in GenBank.The recombinant protein VlpB0was expressed and purified.In addition,the pepfide of the repetitive unit of region Ⅲ was artificially synthesized and conjugated with KLH.Furthermore,vlpB gene sequences encoding 3, 6,9and 12times of repetitive unit in region Ⅲ were artificially synthesized and inserted into pET-32a(+),respectively.The corresponding recombinant proteins VlpB3/6/9/12were prepared.After that,all the recombinant proteins,as well as the conjugated protein,were detected for their adherence to host cell using the microtiter plate adherence assay.The result indicated that the recombinant VlpB0only containing region Ⅱ,as well as the conjugated protein KLH-PepVlpB containing repetitive unit in region Ⅲ,was able to bind host cell.The cytoadhesive capability of VlpB was associated with the times of repetitive unit in region Ⅲ. As the repeated times increased,the adherence significantly decreased.In conclusion,both the region Ⅱ and Ⅲ of vlpB contain cytoadhesion sites,while the cytoadhesive ability of the whole molecule decreased as the times of repetitive unit in region Ⅲ increased.The results of this study laid the foundation for further study on the pathogenic mechanism of M.hyorhinis.