RECOMBINANT EUKARYOTIC EXPRESSION PLASMID ENCODING pprI GENE OF DEINOCOCCUS RADIODURANS R1 AND ITS FUNCTIONS

A method for constructing the pCMV-HA-pprI eukaryotic vector capable of expressing the pprI gene of Deinococcus radiodurans R1, comprising:

• (1) Cloning the pprI gene, comprising isolating Total genomic DNA from D. radiodurnas R1 as a template of PCR-amplification, the pprI gene clone primers was designed according to the promulgated genome sequence of D. radiodurans R1, wherein the forward primer is 5′-ATGCCCAGTGCCAACGTCAGCCCCCCTT-3, the reverse primer is 5′-TCACTGTGCAGCGTCCTGCGGCTCGTCC-3′, and wherein PCR was carried out with the above template and primers, and PCR products were purified using an agarose gel recovery and purification kit, detected and quantified by agarose gel electrophoresis;
• (2) The construction of a sub-cloning vector pGEM-T, comprising ligating the PCR product pprI gene into a sub-cloning vector pGEM-T, the ligated product pGEM-T-pprI being transferred into E. coli DH5α, and the positive clones picked out after incubation of the bacteria, then the pGEM-T-pprI extracted and sequenced;
• (3) The construction of recombinant plasmid pCMV-HA-pprI, comprising amplificating the recombinant vector pGEM-T-pprI using PCR with the two PCR primers, 5′-TCGAATTCCCAGTGCCAACGTCAGCCCCCCTTGC-3′ and 5′-TTCTCGAGTTTCACTGTGCAGCGTCCTGCGGCTC-3′, the underlined sequences being restriction sites of EcoRI and XhoI respectively, the PCR product digested by EcoRI and XhoI, and the fragment ligated into the pCMV-HA vector that had been predigested by the above enzymes, and wherein the recombinant plasmid pCMV-HA-pprI is transferred into E. coli DH5α, and cultured on LB plates solidified with 1.5% agar and supplemented with 100 μg/ml of ampicillin, the positive clones picked out after 12 h of incubation, and wherein PCR-amplification is carried out from different positive clones, the clones which contain recombinant plasmid pCMV-HA-pprI selected out by agarose gel electrophoresis, the recombinant plasmid pCMV-HA-pprI separated and sequenced, the positive bacterial clones having the correct sequence conserved.