PGBKT7-hSOD1cDNA Recombinant Plasmid And Sequence Analysis

Objective (1) Application of molecular biology techniques, from normal human liver cells amplified hSOD1cDNA;(2)Using gene technology, build a pGBKT7-hSOD1cDNA plasmid; (3) The double digested gel electrophoresis and sequencing for the experimental results and report on the sequencing results hSOD1 gene sequence and structure related bioinformatics analysis, for understanding the structure and function of the gene for reference.Methods (1)The test cells extracted from human liver total RNA,and then reverse transcriptase it first chain.According to GeneBank human Cu/Zn-SODcDNA sequence (serial number AB087266)a pair of PCR primers,generated after reverse transcription of the first PCR amplification of cDNA chain,obtained gene .(2) Construction and identification of shuttle plasmid:â‘ Recovery stored in our laboratory shuttle plasmid pGBKT7 containing the DH5É‘bacteria extracted using plasmid extraction kit plasmid pGBKT7;â‘¡with EcoRâ… , Salâ… digested plasmid pGBKT7, pGBKT7 to obtain linearized fragment in the role of T4DNA ligase gene connected with the recombinant plasmid pGBKT7-hSOD1cDNA;â‘¢The recombinant plasmid pGBKT7-hSOD1cDNA for restriction enzyme digestion;â‘£The recombinant plasmids were sent to sequencing, DNA sequence analysis of the company. (3) hSOD1cDNA genetic analysis of biological information: According to the report sequencing results hSOD1 gene sequence, using bioinformatics its homology to other items of the analysis and prediction of molecular weight.Results (1) the use of molecular biology techniques, successfully amplified hSOD1cDNA genes. (2) plasmid pGBKT7-hSOD1cDNA after EcoRâ… , Salâ… double digested, further analysis of recombinants were sequenced to verify the sequence of the cloned hSOD1cDNA in GeneBank (AB087266) announced hSOD1 sequences match. The results showed that the recombinant pGBKT7-hSOD1cDNA inserted gene is positive, single-fold insert. (3) hSOD1 bioinformatics analysis showed that, hSOD1 length of 459bp, encoding 153 amino acids, through the GeneBank with human SOD1 gene query found 99% sequence homology.Conclution (1) the recombinant plasmid pGBKT7-hSOD1cDNA, for further study of hSOD1 protein function and protein interacting with it laid material foundation for the network; (2) analysis of hSOD1 gene sequence of the gene contribute to further understanding of the molecular and biological functions.