论文部分内容阅读
目的将含有抗人乙酰胆碱受体(AChR)单链抗体基因的载体重新构建并建立表达菌株。方法采用聚合酶链反应(PCR)从质粒pHEN1中克隆单链抗体scFv1929#全长基因,将PCR产物再定向克隆至载体pET32a(+)的多克隆位点中,并转入大肠杆菌BL21(D E3)plysS以构建表达菌株。结果PCR产物约730bp,与预期一致,重组阳性克隆菌株JM109+pET32a(+)-scFv1929#经菌液PCR可见730 bp处有特异性条带,所提取质粒pET32a(+)-scFv1929#经PCR及酶切鉴定正确,经测序后证实scFv1929#的核苷酸序列正确并正确地插入载体pET32a(+)的读码框内。将新构建载体转化大肠杆菌BL21(D E3)plysS以获得表达菌株。结论已成功地重新构建含有抗AChR单链抗体基因的载体并建立大肠杆菌表达菌株BL21(D E3)plysS+pET32a(+)-scFv1929#,为今后表达抗AChR单链抗体scFv1929#融合蛋白打下基础。
OBJECTIVE: To reconstitute vector containing anti-human acetylcholine receptor (AChR) single-chain antibody gene and to establish expression strain. Methods The full-length single-chain scFv1929 # gene was cloned from the plasmid pHEN1 by polymerase chain reaction (PCR). The PCR product was cloned into the multi-cloning site of vector pET32a (+) and transformed into E. coli BL21 (D E3) plysS to construct the expression strain. Results The PCR product was approximately 730bp. As expected, the recombinant positive clones JM109 + pET32a (+) - scFv1929 # showed a specific band of 730 bp by PCR. The pET32a (+) - scFv1929 # After restriction analysis, the nucleotide sequence of scFv1929 # was confirmed to be correctly and correctly inserted into the reading frame of vector pET32a (+). The newly constructed vector was transformed into E. coli BL21 (D E3) plysS to obtain the expression strain. Conclusion The vector containing anti-AChR scFv1929 # fusion protein has been successfully reconstructed and the E. coli expression strain BL21 (D E3) plysS + pET32a (+) - scFv1929 # has been successfully constructed. .