The Research For The Construction And Expression Of New-type RPA(K) Prokaryotic Vector

In order to reduce the cost of the research, make its expression, quantitation and purification easier, rPA (K) cDNA was cloned into prokaryotic expressive vector pET-30a (+), its expression was optimized and it was renatured and purified. The results were:1. By using PCR, we got the rPA (K) cDNA, then it was cloned into pET-30a (+) to get the new-type rPA (K) prokaryotic vector and it was expressed successfully. The content of target protein went to 24.3% and the average density was 0.16. The molecular weight was about 43 kD. The ELISA indicated the expressive product had specific positive response with anti-t-PA antibody and its average content was 210ng/mL.2. We optimized some factors influencing the product's expression through Uniform Design. The best expressive conditions were: dissolved oxygen was 90%, IPTG inducing time was 5.3 h, pH was 6.0, final concentration of IPTG was 0.1 mmol/L, and the culture was HD. The result showed the average density increased from 0.16 to 0.48, so the yield of expression had been improved 3 times.3. Different kinds of detergent (Urea, Triton X-100, and ethanol) were used to wash the inclusion body by turns and ammonium sulfate precipitation was used last. The result showed that the purity of rPA(K) inclusion body was increased from 16.0% to 56.6% by 2 M urea, 0.1% Triton X-100,20% alcohol and 15% (NH₄)₂SO₄.4. The dialysis renaturation and dilution renaturation was used to the renaruration of target product respectively. Through dialysis renaturation, the specific activity was 1.33×10⁵ IU/mg; while it was 1.06×10⁵ IU/mg by using dilution renaturation. The former was as 1.25 times as the later, so the former was better.5. S·Tag^TMPurification Kit was used in the purification experiment and the two kinds of eluent (rEK and MgCl₂) were compared. SDS-PAGE showed that the molecular weight of target protein had no change by using MgCl₂ and was still 43 kD. While, by using rEK, the purity of product reached to 72% and the molecular weight of it was 40 kD. That meant the non-target fragment was cut off.