F_(212-489)-3LysM融合蛋白与乳酸乳球菌的体外混合展示

The Fusion Protein F_(212-489)-3LysM Displayed on the Surface of Lactococcus lactis In vitro

探索C端融合有锚定序列3Lys M的呼吸道合胞病毒F截短蛋白(F212-489),能否通过体外混合展示于乳酸乳球菌MG1363表面。采用PCR方法扩增f212-489及3lys M基因,分别亚克隆至p MD18-T载体,在亚克隆载体中酶切回收上述两片段,插入p ET28a构成p ET28a-f212-489-3lys M,酶切鉴定并测序正确后转化BL21(DE3),经IPTG诱导获得的包涵体蛋白进行溶解、复性,将复性后的F212-489-3Lys M与乳酸乳球菌MG1363体外混合,通过全菌ELISA(enzyme linked immuno sorbent assay)检测融合蛋白吸附菌体的情况。成功构建重组菌p ET28-f212-489-3lys M/BL21(DE3),该菌诱导后F212-489-3Lys M主要以包涵体形式表达,F212-489-3Lys M与MG1363混合后,经全菌ELISA检测,F212-489-3Lys M组OD450远高于对照组,且差异非常显著(P<0.01)。证明经大肠杆菌表达的重组蛋白F212-489-3Lys M经体外混合可展示于乳酸乳球菌MG1363表面。

To explore whether the exogenous protein F212-489 containing the membrane anchored sequence could be displayed on the surface of Lactococcus lactis MG1363. The exogenous gene and anchor sequence were amplified by PCR and then inserted into the p MD18 T vector respectively. The two fragments cut from the sub-clone vectors were purified and inserted into p ET28 a to construct p ET28a-f212-489-3lys M. The confirmed plasmid was transformed into BL21（DE3）. Protein from inclusion body induced by IPTG was dissolved and renaturated, and further used for the mixed display in vitro. The whole-bacterium ELISA（enzyme linked immuno sorbent assay） was used to detect the adsorption condition between fusion protein and MG1363.p ET28-f212-489-3lys M/BL21（DE3） was successfully constructed. The fusion protein was mainly induced as inclusion body. The OD450 of F212-489-3lysm group is much higher than the control group with significant differ-ence（P0.01） was detected by the whole-bacterium ELISA. It proved that the fusion protein from E.coli can be displayed on the surface of Lactococcus lactis MG1363 in vitro.