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@#Objective To express and purify the E protein Domain Ⅲ(ED Ⅲ) of tick-borne encephalitis virus(TBEV) in tandem and prepare the corresponding polyclonal antibody.Methods The TBEV RNA was extracted by Trizol method,and then reversely transcribed into cDNA,which was used as template to amplify ED Ⅲ gene fragment by PCR.Two ED Ⅲ gene fragments were ligated into fusion gene by the hydrophobic flexible polypeptide(G_4S)_3 using overlapping PCR,which was then linked to prokaryotic expression vector pET-28a(+) to construct the recombinant expression plasmid pET-28a-2ED Ⅲ.After sequencing,pET-28a-2ED Ⅲ was transformed into E.coli BL21(DE3) competent cells,induced by IPTG and purified by Ni~(2+) affinity chromatography.Female New Zealand white rabbits were immunized with the renatured recombinant protein to prepare polyclonal antibody.The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot.The homology of ED Ⅲ amino acid sequence between TBEV and other flaviviruses was analyzed by DNAMAN software.Results The recombinant plasmid pET-28a-2ED Ⅲ was identified by sequencing,and the amplified sequence contained two genes consistent with the E sequence of TBEV "Senzhang" strain(JQ650523.1) included on GenBank,indicating that the recombinant plasmid was constructed correctly.The recombinant 2ED Ⅲ protein was expressed mainly in the form of inclusion bodies,with a relative molecular mass of about 21 000 and a purity of 97.5%.The titer of rabbit anti-2ED Ⅲ serum polyclonal antibody was 1:10~7,which reacted specifically with TBEV whole virus.DNAMAN software alignment showed that the homology of ED Ⅲ amino acid sequences between TBEV and Japanese encephalitis virus(JEV),yellow fever virus(YFV) and Dengue virus(DENY) was 36.56%,9.28% and 30.77%,respectively.Conclusion The TBEV envelope ED Ⅲ tandem recombinant expression plasmid pET-28a-2ED Ⅲ was successfully constructed.The expressed recombinant 2ED Ⅲ protein had good reactivity and immunogenicity,and the prepared polyclonal antibody had high titer.
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Antibacterial peptide can be easily degraded by protease and has the lethal effect on the host Escherichia coli. In order to solve these problems and further improve the expression ability of the Escherichia coli system, the antimicrobial peptide Spinosan-C of Paa spinosa was studied. First, the codon of Spinosan-C was optimized according to E. coli codon usage frequency. Then, the 8 multimeric Spinosan-C gene (8×Spinosan-C) was synthesized and cloned into prokaryotic expression vector pET-28a. The fusion antimicrobial peptide 8×Spinosan-C was further highly expressed in Escherichia coli strain Rosetta. The recombinant 8×Spinosan-C protein was then purified and cleaved specially by formic acid to generate the Spinosan-C monomer. Antibacterial test in vitro suggested that the cleaved Spinosan-C monomer had antibacterial bioactivity against the test bacteria. This study provides a technical reference for the largescale preparation of frog antimicrobial peptides.
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In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.