猪鼻支原体表面可变脂蛋白vlpA黏附宿主细胞功能研究

doi:10.11843/j.issn.0366-6964.2016.09.019

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (9): 1897-1904.doi: 10.11843/j.issn.0366-6964.2016.09.019

猪鼻支原体表面可变脂蛋白vlpA黏附宿主细胞功能研究

张必雄^1，2，熊祺琰^1* ，王佳¹，纪燕¹，倪博¹，韦艳娜¹，冯志新¹，刘茂军¹，邵国青^1,3*

（1.江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心，南京210014；2山西农业大学动物科技学院，太谷 0308003;3.江苏省肉类生产与加工质量安全控制协同创新中心，南京 210095）

收稿日期:2016-04-01 出版日期:2016-09-23 发布日期:2016-09-23
通讯作者: 邵国青，研究员，Tel:025-84391973，E-mail:gqshaojaas@163.com；熊祺琰，副研究员，Tel:025-84390880，E-mail:qiyanxiongnj@163.com
作者简介:张必雄（1989-），男，湖北天门人，硕士生，主要从事免疫化学与分子免疫学研究，E-mail：zhangbixiongadc@163.com
基金资助:
国家自然科学基金项目（31300155）；江苏省自然科学基金项目（BK20130702）

The Function of the Variable Lipoprotein A of Mycoplasma hyorhinis in Adherence to Host Cell

ZHANG Bi-xiong^1，2，XIONG Qi-yan ^1* ， WANG Jia¹，JI Yan ¹，NI Bo¹，WEI Yan-na¹，FENG Zhi-xin ¹，LIU Mao-jun ¹，SHAO Guo-qing ^1，3*

(1.Institute of Veterinary Medicine，Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-products，Nanjing 210014，China；2.College of Animal Science and Veterinary Medicine，Shanxi Agricultural University，Taigu 030800，China；3.Jiangsu Collaborative Innovation Center for Meat Production，Processing and Quality Control，Nanjing 210095，China）

Received:2016-04-01 Online:2016-09-23 Published:2016-09-23

摘要/Abstract

摘要：

猪鼻支原体（Mycoplasma hyorhinis）可引起猪的多发性浆膜炎、关节炎、肺炎及中耳炎等多种慢性炎症，感染率极高，且与人类肿瘤的发生有关，对人类健康构成威胁。已有研究表明猪鼻支原体表面可变脂蛋白（vlp）家族参与支原体黏附宿主细胞的过程，本试验主要详细研究vlp家族成员之一vlpA的黏附细胞功能，特别是其Ⅲ区重复片段重复次数变化对其黏附能力的影响。根据GenBank中已公布的vlpA的基因序列，设计引物，从菌体DNA中扩增获得vlpA基因，或者人工合成Ⅲ区重复片段重复次数不等的一系列vlpA基因，均克隆至pET-32a(+)质粒中进行表达，获得目的蛋白质。同时利用固相合成法制备含两段Ⅲ区重复片段的多肽。利用间接免疫荧光试验和微孔板黏附试验检测所制备的各种重组vlpA蛋白和多肽的黏附功能。通过诱导表达和镍柱亲和层析纯化，成功获得纯度较高的各个目的蛋白质。利用间接免疫荧光试验证实vlpA可以黏附宿主细胞；利用微孔板黏附试验定量检测不含Ⅲ区的重组vlpA0蛋白及Ⅲ区重复片段多肽的黏附能力，结果发现vlpA0可黏附细胞，而Ⅲ区重复片段多肽无明显黏附能力；进一步利用微孔板黏附试验检测含Ⅲ区重复片段次数分别为3、6、9、12次的重组蛋白vlpA3、vlpA6、vlpA9、vlpA12的黏附能力，结果显示四种重组蛋白质均可黏附细胞，但黏附水平均低于不含Ⅲ区的重组vlpA0蛋白，且随着Ⅲ区重复片段重复次数的增加黏附能力下降。本研究结果提示vlpA为猪鼻支原体黏附因子之一，其Ⅱ区含有黏附位点，而整体分子的黏附能力随着Ⅲ区重复片段重复次数的增加明显减弱。本研究结果有助于进一步研究猪鼻支原体的黏附及致病机制。

Abstract:

Mycoplasma hyorhinis (M.hyorhinis) is a swine pathogen that can cause multiple chronic inflammation including polyserositis，pneumonia，arthritis and otitis media.It is also a potential pathogen for human beings，associated with various human cancers.In previous study，vlp family has been revealed to play a role in mediating cytoadhesion of M.hyorhinis.Herein，we furtherly studied the cytoadhesion function of vlpA，one of the vlp family member，particularly how the repeat time of repetitive unit impact the function.According to the sequence published on GenBank，the vlpA gene was amplified from bacterial genome.Furthermore，vlpA genes containing 0，3，6，9 and 12 times of repetitive unit in region Ⅲ were artificially synthesized.The genes were inserted into pET-32a(+) and expressed in Escherichia coli.At the same time，a peptide containing 2 times of repetitive unit in region Ⅲ was artificially synthesized.All the recombinant proteins，as well as the peptide were detected for their adherence to PK-15 cell by using the indirect immunofluorescence assay and microtiter plate adherence assay.All the recombinant proteins have been successfully induced for expression and purified by affinity chromatography.Adherence of vlpA to PK-15 cell was observed by the indirect immunofluorescence assay.vlpA0 could bind PK-15 cell in the microtiter plate adherence assay，but not the peptide containing 2 times of repetitive unit in region Ⅲ.Furthermore，we detected the adherence of recombinant proteins vlpA3，vlpA6，vlpA9，vlpA12 containing 3，6，9 and 12 times of repetitive unit in region Ⅲ by using the microtiter plate adherence assay，the results showed that all of the four recombinant proteins could adhere to the cell，but the adherence was lower than the recombinant protein vlpA0.Additionally，as the repeat times increased，the adherence significantly decreased.The results of this study suggest that vlpA is one of the adhesion factors of M.hyorhinis.The region Ⅱof VlpA contains cytoadhesion site.The adhesion ability of the whole molecule significantly 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.

中图分类号:

S852.62

张必雄，熊祺琰，王佳，纪燕，倪博，韦艳娜，冯志新，刘茂军，邵国青. 猪鼻支原体表面可变脂蛋白vlpA黏附宿主细胞功能研究[J]. 畜牧兽医学报, 2016, 47(9): 1897-1904.

ZHANG Bi-xiong，XIONG Qi-yan，WANG Jia，JI Yan，NI Bo，WEI Yan-na，FENG Zhi-xin，LIU Mao-jun，SHAO Guo-qing. The Function of the Variable Lipoprotein A of Mycoplasma hyorhinis in Adherence to Host Cell[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(9): 1897-1904.

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猪鼻支原体表面可变脂蛋白vlpA黏附宿主细胞功能研究

The Function of the Variable Lipoprotein A of Mycoplasma hyorhinis in Adherence to Host Cell

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