The Study Of Gene Clone And Expression Of Porcine Resistin, Visfatin And Preparation Of Porcine Resistin Polyantibodies Against Resistin Recombinant Protein

Resistin and visfatin are two adipocytokines found recently. They are correlated to obesity,fat formation and metabolic syndrome mechanism of fatty tissue. Landrace are used and resesitin and visfatin genes are cloned and expressed, then resistin polyclonal antibodies are prepared and visfatin eukaryotic expression vector are constructed. All the work we did are basises for studying the lipogenesis mechanism in swine and pork type selective breeding, and they can also be references for mechanism of resistin and visfatin on metabolic syndrome and medicine development on treatment of metabolic syndrome diseases. The results are as follow:1. Sequence cloning and analysis: According to GenBank, the 360 bp resistin coding sequences and 1.5 kb visfatin coding sequences are cloned from cDNA library of mesentery fat tissue in Landrace by RT-PCR, and the sequences are submission to NCBI (EU707339). According to analysis, we find that the resistin coding sequences of Landrace and the Mei Shan porcine are the same, and the visfatin CDS coding sequences differ in the 47 base and 115 base in visfatin CDS.2. Construction of resistin prokaryotic expression vector pGEX-KG-Rest and high performance expression conditions: According to cloned resistin sequence, designed the primer, amplification mature peptide coding region without signal peptide. and cloned into pGEX-KG. The expression conditions of recombinant prokaryotic expression vector are optimized by different inducing temperatures, inducing time and different LB pH, E. coli, IPTG concentrations. We found that in Rosetta when IPTG concentrations reach to 0.3 mmol/L, inducing time to 3 h, the recombinant protein's expression level didn't increase. The recombinant protein's expression level by 30℃induction is higher than by 37°C induction; Rosetta strain is better than BL21 strain; LB pH6.5 or pH7.8 are better than LB pH6.5 with 30℃.3. Preparation of rabbit anti-porcine polyclonal antibodies: After the recombinant protein is high expressed, inclusion bodies are extracted, and the recombinant protein is purified, then mixed with Freund's adjuvant and was used as the antigen to immunize New Zealand rabbit. After the third injection, there is high antibody valence (1:16), suggesting there are much rabbit anti-porcine polyclonal antibodies.4. Construction of porcine visfatin prokaryotic expression vector pGEX-KG-Vis and high performance expression conditions: According to cloned resistin sequences, primers are designed, amplified, and cloned into pGEX-KG. The expression conditions of recombinant prokaryotic expression vector are optimized by different inducing temperatures, inducing time and different LB pH, E. coli, IPTG concentrations. We found that in Rosetta when IPTG concentrations reach to 0.1 mmol/L, inducing time to 2 h, the recombinant protein's expression level didn't increase any more. The expression of Rosetta strain is higher than BL21 strain.5. Construction of porcine visfatin eukaryon expression vector pcDNA3.1(+)-Vis: According to cloned visfatin sequences, primer are designed, amplified, and cloned into pcDNA3.1(+). The recombinant eukaryon expression vector pcDNA3.1(+)-Vis wasconstructed successfully after examination.