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目的对猪囊尾蚴AF239799-1膜联蛋白基因进行高效原核表达,对表达产物进行免疫学分析。方法通过DNA自动合成仪,用固相亚磷酸酰胺法合成一段5’端有NdeⅠ内切酶位点,3’端有XhoⅠ内切酶位点的猪囊尾蚴AF239799-1基因序列,并将其克隆到原核表达载体pET28a(+)中,经测序、PCR及双酶切鉴定后,将其转入到宿主菌E.coli BL21(DE3)中,用IPTG诱导表达,表达产物采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定,并用Ni-NTA亲和层析柱纯化,纯化的重组蛋白用Western blot进行免疫学分析。结果该基因全长为1 250bp,编码区为2~1 042bp,编码346个氨基酸,理论分子质量、等电点分别为40.39ku和6.80。经DNA测序、PCR及双酶切鉴定,pET28a(+)-AF239799-1重组质粒构建成功。重组质粒在E.coli BL21(DE3)主要以包涵体形式表达,Western blot显示重组蛋白可被猪带绦虫病猪血清识别。结论猪囊尾蚴AF239799-1基因可在原核表达系统中高效表达,表达产物具有免疫反应性。
OBJECTIVE: To efficiently prokaryotic express the AF239799-1 annexin gene of Cysticercus cellulosae and to analyze its expression product by immunology. Methods A DNA fragment of AF239799-1 gene with a NdeⅠ endonuclease site at the 5 ’end and a Xho Ⅰ endonuclease site at the 3’ end was synthesized by a solid phase phosphite method using a DNA automated synthesizer. And cloned into prokaryotic expression vector pET28a (+). After sequencing, PCR and double enzyme digestion, it was transformed into host strain E. coli BL21 (DE3) and induced with IPTG. The expressed product was expressed in dodecyl Sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and purified by Ni-NTA affinity chromatography. The purified recombinant protein was analyzed by Western blot. Results The full-length cDNA of this gene was 1 250 bp with a coding region of 2 ~ 1 042 bp encoding a protein of 346 amino acids with a theoretical molecular mass of 40.39 ku and an isoelectric point of 6.80. After DNA sequencing, PCR and double enzyme digestion, pET28a (+) - AF239799-1 recombinant plasmid was successfully constructed. Recombinant plasmids were mainly expressed as inclusion bodies in E. coli BL21 (DE3), and Western blot showed that the recombinant proteins could be recognized by pig serums of Taenia saginata. Conclusion AF239799-1 gene of Cysticercus cellulosae can be highly expressed in prokaryotic expression system and the expressed product is immunoreactive.