Expression and antibody preparation of recombinant truncated glycoprotein of Guertu virus
10.3760/cma.j.cn112309-20190727-00231
- VernacularTitle:古尔图病毒重组截短糖蛋白基因的表达及抗体的制备
- Author:
Abula AYIPAIRI
1
;
Shu SHEN
;
Jingyuan ZHANG
;
Xijia LIU
;
Yijie LI
;
Fei DENG
;
Yujiang ZHANG
;
Surong SUN
Author Information
1. 新疆大学生命科学与技术学院，新疆生物资源基因工程重点实验室，乌鲁木齐 830046
- From:
Chinese Journal of Microbiology and Immunology
2020;40(3):178-184
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To express truncated glycoprotein (Gn, Gn1, Gn2, Gn3, Gc1 and Gc2) of Guertu virus (GTV) in Escherichia coli ( E. coli) cells, and prepare polyclonal antibodies against recombinant proteins Gn-His, Gc1-His and Gc2-His after purification. Methods:Gene fragments encoding Gn, Gn1, Gn2, Gn3, Gc1 and Gc2 of GTV DXM strain were amplified by RT-PCR, and cloned into the prokaryotic expression vector pET-32a (+ ) to construct recombinant expression plasmids. The transformed E. coli BL21(DE3) strains carrying expression plasmids were induced by IPTG to express target proteins, which were identified by SDS-PAGE. Recombinant proteins Gn-His, Gc1-His and Gc2-His were purified by nickel affinity chromatography and detected by Western blot using GTV-positive sheep serum for analysis of their antigenicity. New Zealand white rabbits were immunized with the purified recombinant proteins. The titers and specificity of serum antibodies were analyzed by ELISA. Meanwhile, eukaryotic expression vectors pcDNA3.1-Gn, pcDNA3.1-Gc1/Gc2 were constructed and transfected into mammalian Vero cells to evaluate the binding activity of rabbit polyclonal antibodies by indirect immunofluorescence method. The specific reactivity of serum antibodies to recombinant proteins was detected by Western blot. Results:Restriction enzyme analysis and DNA sequencing confirmed that the recombinant expression vectors of pET-32a-Gn, pET-32a-Gn1/Gn2/Gn3, pET-32a-Gc1/Gc2, pcDNA3.1-Gn and pcDNA3.1-Gc1/Gc2 were constructed successfully. The relative molecular mass ( Mr) of the expressed recombinant proteins Gn-His, Gn1/Gn2/Gn3-His, Gc1/Gc2-His were approximately 63.4×10 3, 37.1×10 3, 31.9×10 3, 30.8×10 3, 40×10 3 and 54.4×10 3, respectively. The recombinant proteins could be recognized by GTV-positive sheep serum. The titers of polyclonal antibodies against GTV Gn, Gc1 and Gc2 were 1∶409 600, 1∶204 800 and 1∶6 400, respectively. Indirect immunofluorescence assay and Western blot showed that the prepared rabbit polyclonal antibodies could specifically react with the proteins expressed in eukaryotic cells and the recombinant proteins. Conclusions:The recombinant GTV glycoproteins Gn-His and Gc1/Gc2-His were efficiently expressed and purified and characterized with good immunity. The prepared polyclonal antibodies had high titers and good specificity. This study provided reference for further studying the biological function and detection methods of GTV glycoproteins and research on vaccines.
