摘要
利用PCR技术从血清型1／2a的产单核细胞李斯特菌Lm-4株中扩增出actA基因，经克隆筛选和测序鉴定后，构建成该基因的原核表达载体pGEX-6P-1-actA及pET-actA，转入E．coli后，IPTG诱导目的蛋白的表达。SDS-PAGE结果表明，actA基因在两种载体中均获得表达，融合蛋白的大小分别约为120kDa和97kDa。以纯化蛋白为材料进行了AetA单抗的研制，获得4株抗ActA的单克隆抗体杂交瘤细胞株，腹水单抗ELISA效价为1：5×10^4-1：1×10^5。选取单抗1A5进行Western blot分析，结果表明单抗1A5能和表达产物进行特异性反应，且与Lm-4多抗血清的Western blot结果一致。actA基因的原核表达及单抗的研制为研究ActA蛋白的生物学活性及其致病作用奠定了基础。
The actA gene was amplified from Lm-4 strain of Listeria monocytogenes serotype 1/2a by PCR and inserted into T vector. Sequencing showed actA gene was 1833bp long and nucleotide homology was 100% compared with actA gene of Listeria monocytogenes EGD strain in GenBank. The cloned actA gene was then inserted into prokaryotic expression vector pGEX-6P-1 and pET respectively. The predicted fusion protein was detected by SDS-PAGE after IPTG induction of recombinant bacteria. The fusion protein expressed in both vectors showed approximate molecular weight of 120kDa and 97kDa. The expressed fusion protein His-ActA was purified and used as antigen to immunize BALB/c mice, hybridomas were generated with traditional hybridoma techniques. McAbs were screened by ELISA, four hybridoma cell lines secreting antibodies against ActA protein were established and the ELISA titer of these ascitic McAbs were around 1 ：5 × 10^4 - 1 ： 1 × 10^5 . The subtype and specifity of McAbs were identified by kit and Western blot. The McAb 1A5 reacted with the expressed fusion protein GST-ActA and His-ActA in Western blot, consistent with that of mouse anti-Lm- 4 polyclonal antibodies. The successful expression of ActA protein in E. coli and preparation of its monoclonal antibodies has provided useful tools for studies on the biological activity of ActA protein and its role in listerial pathogenesis .
出处
《微生物学报》
CAS
CSCD
北大核心
2006年第6期999-1002,共4页
Acta Microbiologica Sinica
基金
国家自然科学基金(30425031)
国家"973项目"(2006CB504400)
教育部TRAPOYT(175)
扬州大学科学基金项目(OK0313105)~~
