Prokaryotic soluble expression of recombinant G2 envelope glycoprotein from Hantaan virus strain PS-6 and preparation of its monoclonal antibodies
10.13200/j.cnki.cjb.004776
- VernacularTitle:重组汉滩病毒PS-6株G2包膜糖蛋白原核可溶性表达及其单克隆抗体的制备
- Author:
Xiangtao ZHANG
- Publication Type:Journal Article
- From:
Chinese Journal of Biologicals
2026;39(10):1174-1179
- CountryChina
- Language:Chinese
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Abstract:
Objective To solubly express the recombinant envelope glycoprotein G2 of Hantaan virus(HTNV) strain PS-6 in a prokaryotic system, and prepare its monoclonal antibodies(mAbs), in order to provide high-quality mAbs for antigen detection during related vaccine research and production.Methods The cDNA synthesized by reverse transcription of HTNV PS-6 RNA was used as a template to amplify the G2 gene by PCR, and the maltose-binding protein(MBP) tag gene was amplified by PCR using the plasmid pET-28a(+)-MBP-LA-PTH as the template. Both target genes were inserted into the vector pET-28a(+) via homologous recombination to construct the recombinant expression plasmid pET-28a(+)-MBP-PS-6-G2. The plasmid was transformed into competent E.coli BL21(DE3) cells, and after IPTG-induced expression, soluble recombinant G2 protein was obtained via three-step purification of Ni affinity chromatography → HRV-3C protease cleavage → Ni affinity chromatography. Three female BALB/c mice were immunized with the purified protein, hybridoma clones that stably secrete anti-G2 protein mAbs were screened by hybridoma fusion technology, and ascites was prepared and purified. The purity of mAbs was measured by SDS-PAGE, the specificity was identified by Western blot, and the relative affinity was determined by indirect ELISA.Results Colony PCR and double enzyme(XhoⅠ/NdeⅠ)digestion confirmed that the recombinant expression plasmid was constructed correctly. The expressed product had a relative molecular mass of approximately 95 000 and was expressed in a soluble form in the supernatant. The purified protein exhibited a concentration of 1. 2 mg/mL, a purity of > 95%, and specific binding to HTNV PS-6 mAbs. A total of four hybridoma cell lines, 3C4, 4D6, 2A8 and 5E2, were screened by hybridoma fusion technology, and the titers of ascites were 1∶10~6, 1∶10~6, 1∶10~6 and 1∶10~7, respectively. The purity of antibodies after purification was above 95%, and all of them could specifically recognize recombinant G2 protein and HTNV PS-6 inactivated virus bulk solution, with relative affinities of(0. 090 ± 0. 005),(0. 140 ± 0. 010),(0. 292 ± 0. 008) and(0. 237 ±0. 010) mg/mL, respectively.Conclusion The soluble expression of envelope glycoprotein G2 of HTNV PS-6 in prokaryotic system was successfully achieved by MBP solubilization tag and three-step purification process, and four mAbs with high specificity and affinity were obtained, providing a valuable biological tool for HTNV antigen detection and vaccine research.
