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目的生物工程方法在原核系统内可溶性表达巨细胞病毒gp52蛋白并进行纯化,获得可以用于检测巨细胞病毒特异性抗体的重组抗原。方法利用PCR扩增获得巨细胞病毒gp52核酸序列,然后克隆至pET-DsbC核融合表达载体中,在原核系统进行诱导表达并纯化,用Western blot和ELISA检测其抗原性及实用性。结果表达纯化的DsbC-gp52融合蛋白分别经酶标记建立IgM捕获ELISA方法,检测60份抗巨细胞病毒IgM阳性血清和60份阴性血清。其中用酶标记DsbC-gp52蛋白建立的捕获ELISA法阳性检出率达96.6%,阴性检出率100%。结论融合蛋白DsbC-gp52在大肠杆菌中主要以可溶性表达形式存在,获得的高纯度融合蛋白抗原性和特异性强,利用其建立的IgM捕获ELISA方法,可用于临床检测风疹病毒的早期感染。
Objective The bioengineering method expresses the cytomegalovirus gp52 protein in the prokaryotic system and purifies it to obtain a recombinant antigen that can be used to detect cytomegalovirus specific antibodies. Methods The cytomegalovirus gp52 gene was amplified by PCR, then cloned into pET-DsbC nuclear fusion expression vector, expressed in prokaryotic system and purified. Western blot and ELISA were used to detect the antigenicity and its practicability. Results The purified DsbC-gp52 fusion protein was detected by enzyme-linked immunosorbent assay (ELISA) to detect IgM-positive sera and 60 negative sera. The positive rate of capture ELISA established by enzyme-labeled DsbC-gp52 protein was 96.6%, and the negative detection rate was 100%. Conclusion The fusion protein DsbC-gp52 exists mainly in soluble form in Escherichia coli. The obtained fusion protein of high purity has strong antigenicity and specificity. The established IgM capture ELISA method can be used for the clinical detection of early infection of rubella virus.