LTB-MOMP融合基因表达载体的构建及其在原核细胞中的表达

目的构建含大肠杆菌热不稳定肠毒素B亚单位(LTB)和鹦鹉热衣原体主要外膜蛋白(MOMP)融合基因(LTB-MOMP)的表达载体,并在原核细胞中表达融合蛋白。方法应用PCR从质粒EWD299和鹦鹉热衣原体中扩增LTB和MOMP基因,经与柔性肽连接后,插入pET-28a载体中,酶切鉴定正确后,转化大肠杆菌Rosetta,IPTG诱导表达,并纯化目的蛋白。SDS-PAGE和Westernblot分析外源蛋白的表达及表达产物的抗原特异性。结果重组工程菌可以表达相对分子质量约为54000的LTB-MOMP融合蛋白,表达量占菌体总蛋白的42%,LTB-MOMP融合蛋白具有良好的抗原特异性。结论已成功构建了LTB-MOMP融合基因表达载体,并在原核细胞中获得表达。

Construction of Prokaryotic Expression Vector for LTB-MOMP Fusion Gene

Objective To construct an expression vector for fusion gene of heat-liable enterotoxin B subunit (LTB) and major outer membrane protein(MOMP) of Chlamydia psittaci(CP) and express the fusion gene in prokaryotic cells. Methods Amplify LTB and MOMP genes from EWD299 and CP by PCR respectively and link with a flexible peptide (Gly4Ser)3, then insert into vector pET-28a. Identify the constructed recombinant plasmid by restriction analysis and transform to E. coli Rosetta for expression under induction of IPTG. Purify the expressed protein and identify by SDS-PAGE and Western blot. Results The expressed LTB-MOMP fusion protein, with a relative molecular mass of about 54 000, contained 42% of total somatic protein and showed good antigenic specificity. Conclusion The expression vector for LTB-MOMP fusion gene was successfully constructed and expressed in prokaryotic cells.