Design and construction of cDNA mutant of human sAPRIL / 第三军医大学学报

ABSTRACT

Objective To construct 6 kinds of human soluble proliferation-inducing ligand (sAPRIL) cDNA mutants. Methods Six pair primers according to the cDNA sequence of human sAPRIL for different mutants were designed as follows HEL Th epitope was added at C or N end, the last or first 45 bp DNA was deleted and HEL Th epitope was added, the last or first 90 bp DNA was deleted and HEL Th epitope was added. Different enzyme site was designed at 5′ end of 12 primers, BamH Ⅰ or SalⅠ or HandⅢ. At the same time, 2 pairs of HEL Th epitope sequence were designed and synthesized, and different incision enzyme sticky end sequence was added at the 5′ or 3′ end of each epitope DNA. Mutant sequences were amplified by 6 pairs primers with sAPRIL cloning plasmid as template. The PCR fragment was digested and then ligated with the HEL Th epitope DNA that had been reannealed. The ligation fragments of the head were ligated with digested pQE80 vector fragment again, and were transformed into DH5? competent cell. Recombinant plasmids were identified by digestion and sequencing. Results Six sAPRIL cDNA mutant DNA sequences were obtained by PCR, and the expression plasmids of sAPRIL cDNA mutants including HEL Th epitope sequence were constructed successfully. The result of sequencing proved that mutations generated as designed fully. Conclusion Six sAPRIL cDNA mutant expression plasmids were constructed successfully.