猪鼻支原体表面可变脂蛋白vlp家族介导黏附宿主细胞功能研究

Mycoplasma hyorhinis(Mhr) can induce multiple chronic inflammatory diseases in pigs, and has also been proved to be associated with sereval kinds of human cancers. The adherence to host cell is the very critical step for its infection. However, the adhesion molecule of Mhr is still unclear. Variable lipoproteins(vlp) are the major surface moleculars of Mhr, which provide a highly variable surface feature of Mhr and help it to escape from the host immunity reaction. The result of the preliminary experiment showed that a recombinant protein containing partial fragments of vlp family can adherent to host cell, and its antibody could inhibit the adherence of Mhr to host cell. This indicated the involvement of vlp in the adherence process. The aim of the present project is to study the role of vlp as adhesion molecule. The assay of adherence inhibition by antibody and recombinant protein would be used to comfirm the adhesion function of vlp family. After this, the function of eath vlp member would be identified one by one. The influence of number of the repeated sequence in region III of the identified vlp on its adhesion function would also be detected during this part.Furthermore, the expression of vlp family and the host cell adhesion mediated through vlp would be subquently observed on multiple strains with multiple gernerations.Based on this result, the relationship and change regulation between vlp-mediatied surface immunogenicity variation of Mhr and vlp-mediated host cell adhesion of Mhr could be illuminated. The result of this study would greatly help the pathogenisis research and the vaccine development of Mhr.

猪鼻支原体（Mhr）可引起猪的多种慢性炎症，同时也与多种人类肿瘤发生相关。黏附宿主细胞在Mhr感染致病中为关键步骤，但其黏附因子目前尚不清楚。Mhr表面分布有许多可变脂蛋白（vlp家族），为其实现抗原性变化、免疫逃避的关键因素。前期研究结果显示含vlp部分片段的重组蛋白可黏附细胞，其抗体可对黏附产生抑制，提示vlp参与介导Mhr黏附宿主细胞。本项目拟对vlp家族的黏附功能开展研究。首先通过抗体抑制和蛋白竞争的方法确实证明vlp家族具有介导Mhr黏附细胞的能力，在此基础上分别对7种vlp成员的黏附功能进行逐一鉴定，并分析其重复区重复次数变化是否影响黏附功能。同时，通过观察多菌株多代次Mhr中vlp的表达情况和介导细胞黏附情况，分析阐述vlp介导Mhr表面抗原性高频改变和介导Mhr黏附宿主细胞能力变化之间的关系和规律。对上述问题的阐释将有助于Mhr致病分子机制的深入研究和疫苗产品的开发。

猪鼻支原体可引起猪的多种慢性炎症，同时也与多种人类肿瘤发生相关。黏附宿主细胞在猪鼻支原体感染致病中为关键步骤，但其黏附因子目前尚不清楚。猪鼻支原体表面分布有许多可变脂蛋白（Vlp家族），为其实现抗原性变化、免疫逃避的关键因素。前期研究结果显示含Vlp片段的重组蛋白可黏附细胞，提示Vlp参与介导猪鼻支原体黏附宿主细胞。本项目对Vlp家族的黏附功能开展研究，主要内容和结果包括：（1）通过重组蛋白竞争性抑制猪鼻支原体黏附宿主细胞检测和重组蛋白直接黏附宿主细胞检测，证明了Vlp家族具有介导猪鼻支原体黏附宿主细胞的能力；（2）发现7种Vlp成员均具有黏附细胞能力；（3）主要黏附位点均位于Ⅱ区；VlpB、VlpG的Ⅲ区重复短肽含有黏附表位，其他Vlp的Ⅲ区重复短肽未检测到明显的黏附能力;（4）7种Vlp成员的黏附能力均随着Ⅲ区重复片段重复次数的增加而降低，不含Ⅲ区或Ⅲ区重复次数少时，黏附能力强；反之，Ⅲ区重复次数多时，黏附能力弱。可能重复次数增多时，Ⅲ区变长，遮蔽了位于Ⅱ区的主要黏附位点，导致整体分子黏附细胞能力降低;（5）当猪鼻支原体受到抗体压力时Vlp分子Ⅲ区重复次数增加，这可能与长Vlp分子有助于实现免疫抵抗有关。本研究证明了Vlp具有黏附细胞功能，为国内外第一个报道的猪鼻支原体黏附因子，系统地研究了各个成员的黏附能力，并阐明了其黏附能力随着Ⅲ区重复片段重复次数的增加而降低的共同变化规律，为研究Vlp是如何帮助猪鼻支原体实现免疫逃逸和长期感染提供了新的启示，并对其他支原体的可变蛋白家族的生物学功能研究提供参考。