人PSP94全长cDNA的获得及PSP94-TNF~Δ融合蛋白的构建

Human PSP94 cDNA Obtained and Expression of 5′PSP TNF Δ Fusion Protein in E.coli

利用ＲＴ－ＰＣＲ从人肥大前列腺组织钓取９４个氨基酸的人前列腺分泌蛋白（ＰＳＰ９４）全长ｃＤＮＡ，序列分析结果与文献报道的完全一致．将ＰＳＰ９４成熟肽与人ＴＮＦα衍生物（ＴＮＦΔ）通过Ｌｉｎｋｅｒ－ＳＡＰＧＴＰ在基因水平上融合成５′ＰＳＰ９４－ＴＮＦΔ，融合基因ＤＮＡ序列分析结果与设计的相符合．５′ＰＳＰ９４－ＴＮＦΔ在大肠杆菌中表达产物分子量约为３１ｋＤ，表达量约占菌体总蛋白量的３５％．以Ｌ９２９细胞和人前列腺癌细胞株ＰＣ－３为靶细胞进行细胞毒分析结果表明，５′ＰＳＰ９４－ＴＮＦΔ融合蛋白既具有ＴＮＦ的细胞毒活性。

Human prostate secretory protein of 94 amino acids (PSP94) cDNA including signal and mature peptide has been successfully obtained by reverse transcription polymerase chain reaction (RT PCR) from human hypertrophic prostate mRNA.Sequence analysis indicated that the sequence of human PSP94 cDNA was the same as that of human natural PSP cDNA.The hPSP94 cDNA and human TNFα mutant (TNF Δ) gene were fused by an artificial linker SAPGTP.The chimeric gene coding for 5′PSP SAPGTP TNF Δ fusion protein under control of the phage P RP L promoter was constructed.The expression was assayed by SDS PAGE,which showed that the amount of the expressed the protein was about 35% of total bacterial proteins.Western blot indicated that the molecular weight of 5′PSP SAPGTP TNF Δ was in accordance with the estimated value of 31 kD.Through biological activity analysis,the fusion protein has cytotoxicity activity of L929 and PC 3 cell.