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Prokaryotic and Eukaryotic Expression of OmpK of Vibrio Harveyi

Author: HeChaoJun
Tutor: WuZhiXin；MaoZhiJuan
School: Huazhong Agricultural University
Course: Aquaculture
Keywords: Vibrio harveyi Outer membrane protein OmpK in The prokaryotic expression Eukaryotic expression
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 33
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Abstract

Harvey Vibrio (Vibrio harveyi) mariculture common gram-negative pathogens, its unique outer membrane of the cell surface protein (outer membrane proteins, Omps) maintain a stable structure of bacterial cells material exchange and bacteria plays a very important role in the pathogenesis of some species such as OmpK has good immunogenicity. Vibrio outer membrane protein OmpK of a wide host of phage KVP-40 receptor, which is widespread among various Vibrio, Vibrio cell surface a composition of protein, having opportunity to extensive contact with the outside world, it is there may be one of the surface antigen of Vibrio. Specific primers designed according to the published Vibrio harveyi (Vibrio harveyi) outer membrane protein OmpK of the gene sequence (accession number AY332563) the Vibrio harveyi genomic DNA as a template was amplified by PCR method the OmpK mature peptide gene fragments, the size of about 750bp, to build the cloning vector pMD18T-OmpK, sequencing results show that there is a 99% homology of the gene has been published Vibrio harveyi outer membrane protein OmpK gene sequences preliminary indication of the successful cloning of of the OmpK mature peptide gene fragment, and the sequence in GenBank accession number HQ395754. Recycling OmpK the recombinant plasmid pMD18T-OmpK after BamH Ⅰ and Hind III two restriction endonuclease digestion gene fragments, and this gene was subcloned into the plasmid pET30a and transformed into E. coli BL21 (DE3) competent cells, constructed the prokaryotic expression engineered bacteria E.coli/pET30a-OmpK, recombinant bacteria in IPTG induced inclusion bodies expression of recombinant protein, recombinant protein molecular weight of about 33kD, inclusion bodies were washed repeatedly with EDTA, Triton, low concentrations of urea after the dissolution of the high concentration of urea, and finally through the dialysis of the recombinant protein refolding and renaturation of recombinant protein to immunize New Zealand rabbits prepared rabbit polyclonal anti-OmpK serum. As a template to build good expression vector pET30a-OmpK characteristics pPIC9K secretion expression vector designed a pair of primers, the mature peptide gene fragment was amplified by PCR method, and then connected into pPIC9K plasmid construct the recombinant expression vector pPIC9K -OmpK, recombinant plasmids by colony PCR, plasmid digestion and sequencing, the results show that the constructed successfully. The large number of extraction the recombinant plasmid pPIC9K-OmpK and the parental plasmid pPIC9K, after Sac Ⅰ linearization into Pichia pastoris GS115 competent cells by electrical. The reorganization the transformants GS115/pPIC9K-OmpK and GS115/pPIC9K obtained by Geneticin screening anti 4.0mg/mL G418 high copy sub; most of the transformants Mut; discovered target genes identified by colony PCR plate identification by MM / MD successful integration into the in Pichia yeast genome. Positive transformants secretion under methanol induced expression of recombinant proteins, SDS-PAGE electrophoresis results showed that the molecular weight of the recombinant protein of about 37kD and by excessive glycosylation. The recombinant protein was identified by Western-Blot with prokaryotic expression rabbit anti-OmpK inclusion bodies serum react, the success of recombinant protein expression and immunogenicity is not affected. Expression vector pET30a-OmpK as a template, a pair of primers designed according to the characteristics of the carrier expressed in pPIC3.5K intracellular amplified by PCR OmpK mature peptide gene fragments, and then even into pPIC3.5K plasmid construct a shuttle vector pPIC3 .5 K-OmpK, Sak Ⅰ linearization the recombinant plasmid pPIC3.5K-OmpK and parental plasmid pPIC3.5K after lithium chloride method and transformed into Pichia pastoris GS115 transformants were screened high anti-2.0mg/mL G418 Geneticin Copy sub plate identification of their phenotype by MM / MD, followed by extraction of the genomic DNA of the transformants, two pairs of primers were repeated identification result indicates success constructed yeast the positive transformants GS115/pPIC3.5K-OmpK and the GS115 / pPIC3.5K, positive transformants after methanol induction after intracellular expression of recombinant proteins, SDS-PAGE electrophoresis results showed that the molecular weight of the recombinant protein of approximately 28.7KD, found that the recombinant protein in intracellular inclusion bodies washed glass beads which method expressed, Western-Blot identification of inclusion bodies in serum, rabbit anti OmpK react, indicating successful expression of recombinant proteins in intracellular

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