斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因pET22b载体的构建及表达

CONSTRUCTION OF pET22b VECTOR EXPRESSING THE CYSTEINE PROTEINASE OF ADULT WORM OF PAGUMOGONIMUS SKRJABINI

目的构建含斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因片段的pET22b(+)表达载体,利用SDSPAGE和免疫印迹分析阳性克隆菌株的诱导表达及其表达产物的免疫原性。方法提取斯氏狸殖吸虫成虫总RNA,经RTPCR扩增及T/A克隆后测定其核苷酸序列并进行序列查询与比对。根据已获得的序列和pET22b(+)载体的内切酶位点信息设计引物并再次进行T/A克隆,阳性克隆质粒经NcoⅠ和XhoⅠ双酶切后将目的片段与pET22b(+)载体连接并转化至BL21(DE3)菌株,收集经IPTG诱导表达后的菌液进行SDSPAGE和免疫印迹分析。结果SDSPAGE显示,经诱导表达的阳性克隆菌株、未经诱导菌株与空载体菌株之间的蛋白带型未见明显差异,但经诱导表达的阳性克隆菌株的22ku蛋白条带比另两菌株明显,免疫印迹证实,该蛋白条带可与斯氏狸殖吸虫成虫ES抗原免疫豚鼠血清发生强阳性反应。结论成功构建了含斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因的pET22b(+)载体,经诱导表达的融合蛋白能与斯氏狸殖吸虫成虫ES抗原免疫豚鼠血清发生较强的免疫反应。

Objective To construct pET22b(+) expression vector containing fragments of cysteine protease from adult worm of Pagumogonimus skrjabini, and to analyze the products induced by SDSPAGE and Westen blotting. Methods RNA sequence from adult worm of P. skrjabini was reverse transcripted and amplified to get the cysteine protease cDNA which was cloned into T vector and sequenced. The PCR primers were designed according to obtained partial cDNA sequence of cysteine protease and the sequences from the point of endonuclease in pET22b(+) vector, and amplified to get products of PCR which was cloned into T vector and sequenced for confirmation. To collect the small fragments of products after positive plasmid of T/A clone were hydrolyzed by NcoⅠ and XhoⅠ, and combine to plasmids of pET22b which have been digested by NcoⅠ and XhoⅠ and be transformed into BL21 strain .The products of expression being induced with IPTG were analysed by SDSPAGE and Western blotting. Results There was no apparent difference from their protein bands of SDSPAGE among induced strain, no induced strain and strain without vector, but a band which was molecular weight of about 22 ku from the induced strain was more visible than no induced strain and strain without vector. Furthermore, the strongest immunologic reaction to immune serum of the adult worm of P. skrjabini was examined by Western blotting for the band. Conclusion pET22b(+) expression vector containing fragment of cysteine protease from adult worm of P. skrjabini was successfully constructed.The fusion protein expressed by the strain of positive clone has fine immunoreactive property for immune serum of the adult worm of P. skrjabini.