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为研究恶性疟原虫(Plasmodium falciparum)3D7株AP核酸内切酶(AP endonuclease)的功能,通过PCR的方法扩增PF3D7_0305600(292~1 137 bp),构建克隆载体。测序正确后,将该片段插入到pET-28a和p GEX-4T-1,构建重组表达载体p ET-28a-Ae、p GEX-4T-1-Ae。将表达载体转入BL21-Codon Plus表达重组蛋白r-Ae-His和r-Ae-GST,经SDS-PAGE和Western blot鉴定,r-Ae-His免疫家兔制备多克隆抗体,并用ELISA检测抗血清效价,Western blot检测抗体特异性。结果表明:表达的r-Ae-His分子量约为39 ku,r-Ae-GST分子量约为60 ku。制备的多克隆抗体能够特异性地识别天然蛋白,可用于恶性疟原虫3D7株AP核酸内切酶的后续研究。
To study the function of AP endonuclease of 3D7 strain of Plasmodium falciparum, PF3D7_0305600 (292-1 137 bp) was amplified by PCR to construct a cloning vector. After sequencing, the fragment was inserted into pET-28a and pGEX-4T-1 to construct recombinant expression vectors p ET-28a-Ae and pGEX-4T-1-Ae. The expression vector was transfected into BL21-Codon Plus to express recombinant proteins r-Ae-His and r-Ae-GST. SDS-PAGE and Western blot analysis showed that r-Ae-His immunized rabbits with polyclonal antibody and detected by ELISA Serum titer, Western blot detection of antibody specificity. The results showed that the molecular weight of r-Ae-His was about 39 ku and the molecular weight of r-Ae-GST was about 60 ku. The prepared polyclonal antibody can specifically recognize the native protein and can be used for the subsequent study of Plasmodium falciparum 3D7 AP endonuclease.