Large Scale Expression Of Antimicrobial Peptide CAP18 And Its Antimicrobial Activity Analysis

Antibacterial peptides are an integral part of the host's defense barrier against invading bacteria. They provide immediate protection by direct physicochemical attack on the surface membrane of the microorganisms. Antibacterial peptides are also a most The functional CAP18106-142 was successfully expressed in large scale in E.coli by gene optimization method in this experiment, and detected the antimicrobial activity by bacteriostatic test in vitro and in vivo, which has insignificance in the development of basis of theory and practice to large scale expression CAP18106-142 and the antimicrobial ativity.According to the biased codon used in E. coli, the gene sequence encoding active fragment of CAP18106-142 of mature CAP18 was designed. The gene fragment of c-CAP18 was obtained through PCR-based gene SOEing synthesis method. Multiple copies of c-CAP18 were inserted into the pET-32a prokaryotic expression vector, constructed the fusion expression vectors c-p-CAP18Ⅰ-Ⅷwith 1～8 copies c-CAP18 gene tandem in the same direction and transformed into competent cells of E.coli BL21 for fusion expression. The results showed that the c-CAP18Ⅱfusion protein was achieved by cultivating the E.coli in LB medium at 30°C, inducing the culture when the bacterial cells density reached OD600=1.0 with 0.8mM IPTG, then continuously cultured the cells for 8h.The expression level of soluble c-CAP18Ⅱfusion protein reached 0.788 g/L .In order to choose the medium which fit for large scale expression, use a new complex auto-inducing media wnich was invented by our research group and the expression level of soluble c-CAP18Ⅱfusion protein reached 11.104 g/L which was 14 times higher than in LB whose induction condition had been optimized. The productivity of the c-CAP18Ⅱfusion protein reached 9.788 g/L in 15L fermentor which reached 62.3% of the total soluble proteins. The c-CAP18Ⅱfusion protein was purified by Ni2+ chromatography and was partially cleaved by cyanogen bromide. Choose E.coli, S.typhimurium, L.monocytogenes and S.aures to estimate the antibiosis activity of c-CAP18Ⅱfusion protein in vitro, and use Ampicillin, Kanamycin, Gentamicin and the synthetic c-CAP18 fusion protein as positive control. After 18 hours cultivation the bacterial density of the four bacteria were 0.03525,0.0353,0.07514 and 0.03046 the same as the positive control of the synthetic c-CAP18 fusion protein which bacterial density were 0.02789,0.0271,0.02426 and 0.02968, and had significant differences compared with the negative control which bacterial density were 0.8905,0.8385,0.8 and 0.77 (P<0.01).Choosing swine influenza virus to detect the antivirus activity in vitro, we vaccinated swine influenza virus (H3N2) which was 0.1mL (106EID50) on the spf chicken embryoes. Then we vaccinated the synthetic c-CAP18 and c-CAP18Ⅱfusion protein which was 0.2 mg every embryos after 1 hour at 37°C. After 96 hours we detected the viral titer by hemagglutination method. The result was that the average hemagglutination titer of c-CAP18Ⅱfusion protein was 106.83 the same as he synthetic c-CAP18 fusion protein (106.5) and had significant differences compared with the negative control which was 108.125 (P<0.05).Meanwhile, we use Balb/C mice to detect the antivirus and antibiosis activity. Choose E.coli, S.aures and swine influenza virus (H3N2) and use the synthetic c-CAP18 fusion protein as positive control. We vaccinated swine influenza virus (H3N2) which was 106EID50 every mouse and vaccinated E.coli and S.aures which was 2×106CFU every mouse. Then delivered the the synthetic c-CAP18 fusion protein which was 0.2mg every mouse by nasal to the former and delivered Kanamycin the same dose lasting 3 days. After 1 week we took 2 mice from every group and detected the activity by colony counting method and hemagglutination method. The result was that the higtest bacterial density were 2.41×107 and 1.41×107 the same as the positive control which bacterial density were 1.08×107 and 0.99×107, and had significant differences compared with the negative control which bacterial density were 4.33×107 and 2.08×107 (P<0.05), and the hemagglutination titer of c-CAP18Ⅱfusion protein of heart,hepar,spleen,lung and intestinum crassum were 104.3,103.6,104.3,104.6 and 104.6 the same as he synthetic c-CAP18 fusion protein (103.6,103.3,103.6,104.3 and 104.3) and had significant differences compared with the negative control which were 106.6,106.3,107.3,108.3 and 107.6 (P<0.01). The results above showed that c-CAP18Ⅱfusion protein was expressed successfully both in small scale and large scale, and had antibacterial and antiviral activity after purification and cleavage to the bacteria and virus which we used in the experiment.