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目的本研究根据GenBank中旋毛虫45kDa抗原基因的序列设计引物,采用PCR技术从旋毛虫肌幼虫cDNA文库中扩增靶基因p45cDNA,并克隆到pMD-18T载体,转化至大肠埃希氏菌NovaBlue后测序分析。获得827bp的p45cD-NA。该序列与GenBank中的旋毛虫p45基因序列相比共有1个核苷酸发生改变,二者的同源性为99%,其编码蛋白质由268个氨基酸组成,与45kDa抗原的同源性为99%。将p45cDNA克隆到原核表达载体pET28a并转化至表达菌BL21star(DE3)后,经IPTG诱导表达出约30kDa的重组蛋白。Western-blot检测表明,p45重组蛋白可以被旋毛虫感染猪血清识别,具有良好的抗原性。
In this study, primers were designed according to the sequence of Trichinella spiralis 45 kDa antigen in GenBank. The target gene p45 cDNA was amplified from the cDNA library of Trichinella spiralis larvae by PCR and cloned into pMD-18T vector. After transformation into Escherichia coli NovaBlue Sequencing analysis. The 827 bp p45cD-NA was obtained. This sequence has 1 nucleotide change compared with the Trichinella p45 gene sequence in GenBank. The two sequences share 99% homology and encode 268 amino acids. The sequence has 99% identity with 45kDa antigen %. The p45 cDNA was cloned into the prokaryotic expression vector pET28a and transformed into the expression strain BL21star (DE3). The recombinant protein of about 30 kDa was induced by IPTG. Western-blot showed that p45 recombinant protein can be recognized by trichinella infected pig serum and has good antigenicity.