猪β2m四聚体前体链重组表达载体的构建和表达

Construction and Expression of the Recombinant Expressing Plasmids for the Tetrameric Leader Chain of Swine β 2m

为构建猪β2m轻链四聚体前体链,通过PCR定点突变技术,将猪主要组织相容性复合体Ⅰ类分子轻链β2m羧基端69位苯丙氨酸(Phe)突变为半胱氨酸(Cys),然后将突变体基因插入到pET-28a载体,转化BL21细胞,经诱导后SDS-PAGE检测蛋白质表达情况。结果显示,β2m突变体基因全长297bp,编码98个氨基酸,其中羧基端69位Phe突变为Cys。突变体基因经诱导表达后进行SDS-PAGE检测,结果显示蛋白质表达大小为10.6ku。本研究构建了猪β2m轻链突变体基因重组表达载体,并成功表达了目的蛋白,为今后构建四聚体链奠定基础。

In order to construct the tetrameric leader chain of swine β2m, the light chain of swine β2m was mutated at site of 69 from phenylalanine （Phe） to cysteine （Cys） by using PCR site-directed mutagenesis technique. Then the mutant β2m gene was inserted into pET-28a and transformed into Escherichia coli BL21. After induction, the expression of target protein was detected by SDS-PAGE. The results demonstrated that the mutant β2m was about 297 bp, with coding for 98 amino acids. It was shown that the phenylalanine at site of 69 was mutated to cysteine. After induction, the mutant β2m was expressed successfully as a protein of 10.6 ku detected by SDS-PAGE. It was concluded that the recombinant expression plasmids of the mutant β2m was constructed and target protein was expressed successfully. This study would lay the foundation for construction of tetramer later.