单链抗体与力达霉素融合蛋白在链霉菌中的表达

Expression of scFv-lidamycin fusion protein in Streptomyces globisporus C-1027

目的构建可表达力达霉素(LDM)辅基蛋白基因与抗IV型胶原酶单链抗体(scFv)融合蛋白的重组菌株,获得既具有靶向性、又具有抗肿瘤活性的scFV-LDM。方法以大肠埃希菌-链霉菌穿梭质粒pBS03为基础构建同源双交换质粒pBS-scFv,利用接合转移将该质粒转入球孢链霉菌(S.globisporus)C-1027,根据抗性差异筛选获得重组菌株。用PCR、DNA印迹法对重组菌株进行验证。用SDS-PAGE检测重组菌株中目的融合蛋白的表达。用抑菌圈试验检测重组菌株发酵液的抑菌活性。结果经同源重组得到重组菌株S.globisporusC-1027-scFv。PCR显示重组菌株扩增产物与预期一致,DNA印迹分析显示重组菌株得到与预期吻合的杂交片段,表明scFv-LDM融合蛋白基因已经成功取代LDM辅基蛋白基因。SDS-PAGE结果显示重组菌株不再产生辅基蛋白。抑菌圈试验显示重组菌株发酵液在第5天能够检测到抑菌活性。初步结果表明,重组菌株可产生scFv-LDM融合蛋白。结论成功构建了可产生scFv-LDM融合蛋白且具有分泌活性的重组菌株。这对改造和研发新型单抗导向药物有实际意义。

Objective To construct a recombinant strain that can express fusion protein of lidamycin (LDM) and anti-type Ⅳ collagenase single-chain Fv antibody (scFv), and thus to establish a ScFv-LDM with both taretting efficiency and anti-tumor activity. Methods A homologous double crossover vector pBS-scFv was constructed using the E.coli-Streptomyces shuttle vector pBS03. The pBS-scFv was transferred into Streptomyces globisporus C-1027 by conjugational transfer. A recombinant exconjugant with double crossover was selected for its thiostrepton sensitivity and apramycin resistance. The recombinant strain was confirmed with PCR and Southern blotting. The anti-bacteria activity of the recombinant strain was determined using antibacterial ring test, and the expression of fusion protein was analyzed by SDS-PAGE. Results A recombinant strain S. globisporus C-1027-scFv was obtained. PCR and Southern blotting showed that the apoprotein gene (cagA) was replaced by the scFv-LDM fusion protein gene successfully, which was similar to the anticipated. The SDS-PAGE found no apoprotein produced by the recombinant strain. By antibacterial ring test, anti-bacteria activity was detected in the fermentation broth on the fifth day. These results primarily demonstrated that the recombinant strain could produce scFv-LDM fusion protein. Conclusions A recombinant strain with anti-bacteria activity, which can express scFv-LDM fusion protein, was constructed. It will be significant to the development of new scFv targeting drugs.