抗体信号肽基因的克隆和序列测定及真核表达载体的构建

根据抗体重链与轻链基因的核苷酸序列设计并合成了1对引物,以猪外周血淋巴细胞基因组mRNA为模板,通过RT_PCR方法获得了一大小约85bp的DNA片段,并将其克隆到pGEM_T载体上进行序列测定,测序结果显示,猪抗体信号肽基因核苷酸序列长度为57bp,编码19个氨基酸,核苷酸及推导的氨基酸序列与已发表的抗体信号肽基因序列一致,同时在信号肽基因3’端下游提供可供肽酶切割的窗口和外源基因的克隆位点。然后将信号肽基因亚克隆到真核表达载体pcDNA3.1( )中,成功构建了一含信号肽序列的真核表达载体3.1_SFc,为外源基因在pcDNA3.1( )中的表达与分泌提供了一有效的信号肽,也为研究基因的功能奠定了基础。

Molecular cloning and sequencing of the gene encoding signal peptide for antibody of swine and construction of its eucaryotic expression vector

A fragment about 85 bp was amplified by RT_PCR technique from the genome mRNA of lymphocyte from swine,using primers designed and synthesized according to the gene sequence encoding signal peptide for antibody of swine.The amplifed fragments were cloned into pGEM_T vector and then sequenced.The determination of nucleotide sequence showed that the gene encoding signal peptide for antibody of swine spanned 57 bp,encoding 19 amino acids and the nucleotide and amino acid sequence of the gene cloned was the same as the reported sequences,and in 3'end understream of the gene encoding signal peptide for antibody of swine the signal peptidase cleavage site and cloning sites for foreign gene could be provided by the recombinant plasmid.and then the gene encoding signal peptide for antibody of swine was subcloned into pcDNA3.1( ) and verified by PCR and restriction endonuclease digestion,the recombinant eucaryotic expression vector of the p3.1_SFc was successfully constructed.This result laid foundation for further study of the recombinant eucaryotic expression vector to produce and secrete a foreign protein from living cells and study of the function of gene.