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目的:重组表达抗人乙酰胆碱受体(AChR)单链抗体(scFv)1956#,提高scFv1956#的可溶性表达量。方法:用PCR扩增含抗人AChR scFv1956#的载体pHEN1中的scFv1956#结构基因,并将其插入到原核表达载体pET44a(+)。用重新构建的载体转化E.coli BL21(DE3)pLysS,IPTG诱导表达。用SDS-PAGE来比较更换载体及不同温度和时间对可溶性表达量的影响,并通过Western blot进行鉴定。结果:PCR产物大小为785bp,与预计相符;所构建质粒经测序证实scFv1956#核苷酸序列正确,并正确插入载体pET44a(+)。诱导后得到的可溶性表达量优于原载体pHEN1。结论:载体pET44a(+)表达的端融蛋白NusA可以帮助scFv1956#的正确折叠,从而获得大量可溶性表达。低温长时间诱导有助于提高可溶性表达的量。
OBJECTIVE: To recombinantly express anti-human acetylcholine receptor (AChR) single chain antibody (scFv) 1956 # and to increase the soluble expression of scFv1956 #. Methods: The scFv1956 # structural gene in pHEN1 containing anti-human AChR scFv1956 # was amplified by PCR and inserted into prokaryotic expression vector pET44a (+). E.coli BL21 (DE3) pLysS was transformed with the reconstructed vector and IPTG induced expression. SDS-PAGE was used to compare the effect of changing the vector and the different temperature and time on the amount of soluble expression, and identification by Western blot. Results: The size of PCR product was 785bp, which was in line with the expectation. The constructed plasmid was verified by sequencing and confirmed that the nucleotide sequence of scFv1956 # was correctly inserted into the vector pET44a (+). The soluble expression level after induction was better than the original vector pHEN1. CONCLUSION: NusA, a telomere expression vector pET44a (+), can facilitate the proper folding of scFv1956 #, resulting in a large amount of soluble expression. Induction at a low temperature for a long time helps increase the amount of soluble expression.