| Objective To express the human procalcitonin(PCT)gene in vitro and obtain PCT recombinant proteins in high-purity.To optimize the PCT protein expression and prepare the mice polyclonal antibody.To analyze the main influential factors in clinical PCT test and the optimal storage medium of PCT recombinant protein.Methods:According to human procalcitonin gene sequence on the NCBI,the primers were designed using the Primer Premier 5.0.The full-length sequence was amplified by PCR and prokaryotic expression vector of PCT/p ET-22b(+)was constructed based on a series of molecular biological techniques such as DNA gel recycling,plasmid DNA extraction,genetic connection and enzymatic identification.The protein expression was induced after the conversion of recombinant plasmid to bacterium E.coli BL21.The factors of protein expression were analyzed such as the concentration of inducer(0.2?0.4?0.6?0.8?1.0mmol/L),induction time(4h?8h?12h?16h?20h),induction temperature(22??27??32??37?)and rotate speed(80?120?160?200?250 r/min).The purification of protein mixture was performed with nickel affinity chromatography,then purified protein was identified by Western blotting and colloidal gold techniques.Balb/c mice were immunized after mixing the recombinant protein with the same volume of adjuvant and grind the mixture into the form of emulsion.The blood was collected and serum separated at 7 days after the third time immunization.The titer of mice polyclonal antibody was detected by ELISA method.The results were analyzed in different specimen type,storage time and temperature.PCT recombinant protein was respectively diluted with medium of physiological saline,fetal bovine serum and PBS.Moreover,the results of PCT at 5d,10 d,20d,30 d were tested and statistical analysis was carried on.Results:The result of agarose gel electrophoresis showed that the PCR amplification product was about 350 bp.Homology comparison analysis showed that the PCT gene fragment(348bp)was successfully inserted into p TG19-T vector,without base mutations.Recombinant expression vector of PCT/p ET-22b(+)was digested into fragments of 350 bp and 5 500 bp with Bam H I and Hind?.SDS-PAGE electrophoresis revealed that the PCT recombinant protein existed in soluble form after ultrasonic broken bacteria,Mr about14 KD,and can be obtained with nickel column affinity chromatography purification method.The identification results by Western blotting and PCT colloidal gold method was positive,revealing that recombinant protein contains histidine label(His-tag)and can react with the PCT antibody.SDS-PAGE electrophoresis of the product under different expression conditiona showed that PCT protein expression in the supernatant was higher under induction time 12~20h,induction concentration 0.2~1.0 mmol/L,induction temperature 22?,rotate speed 80~160r/min.When Balb/c mice were immunized with PCT recombinant protein,the serum antibody titer were greater than 1:512 000 at 7 days after the third immunization.PCT values were decreased in the preservation process of blood samples.The difference between PCT values had statistical significance(P<0.05)at 25? plasma tube for 4h(down 4.1%)and whole blood placed for 2h(down 5.4%),at 4? plasma tube for 4h and the whole blood placed 2h.The PCT values had no change for 20 d at 4? when PCT recombinant protein was diluted to the calf serum,better than saline and PBS.Conclusions:The recombinant vector PCT/p TG19-T and PCT/p ET-22b(+)were successfully constructed,and PCT recombinant protein existed in soluble form.It can be obtained with nickel column affinity chromatography purification.The best conditions of inducing expression are as follow: 22?,IPTG concentration 0.2 mmol/L,12 h and160 r/min.PCT recombinant protein has strong immunogenicity,so it is easy to get high titer PCT mice source polyclonal antibody.The preservation time of blood samples about PCT tests is nomore than 4h,the best within 2h and the calf serum is the preferred medium of PCT recombinant protein storage. |
PDF Full Text Request