猪鼻支原体vlp重复区融合蛋白的构建表达及反应原性分析  

Cloning,prokaryotic expression and reactogenicity analysis of a recombinant protein for detecting Mycoplasma hyorhinis

作　　者：熊祺琰[1] 纪燕[1] 刘占军[1] 马庆红[1] 冯志新[1] 刘茂军[1] 白方方[1] 邵国青[1] 

机构地区：[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

出　　处：《中国人兽共患病学报》2013年第9期877-882,共6页Chinese Journal of Zoonoses

基　　金：江苏省农业科技自主创新资金(No.cx(12)1001-05)~~

摘　　要：目的猪鼻支原体(Mhr)是临床猪场常见病原菌之一,同时研究证实其与多种人类肿瘤有关。目前对Mhr的血清学检测尚无有效的方法。本研究以Mhr可变脂蛋白(vlp)家族为对象,设计vlp家族蛋白重复区片段融合蛋白,以作为检测用包被抗原使用。方法根据大肠杆菌的密码子偏嗜性,设计并构建串联表达7种Mhr可变脂蛋白vlpA、B、C、D、E、F、G重复区片段的重组蛋白vlpA-G基因。将该基因片段装入pET-32a(+)载体中转化大肠杆菌,筛选鉴定获得阳性工程菌。IPTG诱导表达,镍柱亲和层析获得纯化的重组蛋白vlpA-G。Western Blot鉴定该重组蛋白与Mhr阳性血清的反应能力,并利用该重组蛋白为包被抗原初步建立间接ELISA方法用于检测猪血清中的Mhr抗体。结果构建表达vlpA-G重组蛋白的基因工程菌,经PCR及DNA测序正确。IPTG诱导后出现明显蛋白条带,经镍柱亲和层析纯化获得vlpA-G重组蛋白。Western Blot试验证明重组蛋白vlpA-G能够和Mhr阳性兔血清产生明显的阳性杂交带;利用重组vlpA-G为包被抗原初步建立间接ELISA检测方法可成功检测Mhr阳性猪血清,证明该重组vlpA-G蛋白具有良好的反应原性。结论本研究构建了重组蛋白vlpA-G并证实该蛋白具有良好的反应原性,可作为Mhr血清学诊断方法研究的候选抗原,同时也为Mhr重组蛋白疫苗提供可选抗原。In the present study, a recombinant protein containing segments of different variable lipoprotein（vlp） of My- coplasma hyorhinis （Mhr） was constructed to be used as antigen for detecting serum antibody. A gene coding for the recombi- nant protein containing repeating region sequences of all the 7 vlp members was designed and optimized for expressing in Esche- richia coli. The gene was inserted into the prokaryotic expression vector pET-32a（＋ ） and transformed into Escherichia coli. PCR and DNA sequencing was used to identify the positive clone. The recombinant vlpA G was induced to express by IPTG and purified by the Ni-column. The interaction between vlpA-G and positive rabbit serum against Mhr was tested by the West ern blot assay. After that, vlpA-G was used as coating antigen to primarily establish an indirect ELISA method to detect the antibody in the serum of swine. The result indicated that the genetic engineering bacteria expressing recombinant vlpA G was successfully constructed confirming by PCR and DNA sequencing. An obvious protein band existed after induction by IPTG. Then vlpA-G protein was purified by using the Ni-column. In the assay of Western blot, the purified vlpA-G showed a strong reaction with the standard positive rabbit serum for Mhr. The vlpA G was then used to establish an indirect ELISA method,and showed positive result when detecting the Mhr positive pig serum. In conclusion, a recombinant vlpA-G protein is constructed and proved to be reactogenic to the antibody {or Mhr. The result of this research provided a potential antigen for developing the diagnostic methods for Mhr, as well as

关 键 词：猪鼻支原体 可变脂蛋白 原核表达 反应原性 

分 类 号：R375[医药卫生—病原生物学]