Prokaryotic expression and purification of nucleoprotein of Guertu virus and its establishment of ELISA detection method
10.3760/cma.j.cn112150-20220326-00286
- VernacularTitle:古尔图病毒核蛋白的原核表达纯化及ELISA检测方法的建立
- Author:
Boyong JIANG
1
;
Jingyuan ZHANG
;
Junzhong WANG
;
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，新疆乌鲁木齐 830046
- Keywords:
Nucleoproteins;
Enzyme-linked immunosorbent assay;
Serological test;
Expression and purification;
Guertu virus
- From:
Chinese Journal of Preventive Medicine
2022;56(6):824-830
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To obtain purified protein antigen of guertu virus (GTV) nucleoprotein (NP) and establish a rapid and accurate enzyme-linked immunosorbent assay (ELISA) method for detection of GTV antibody.Methods:Codon optimized GTV NP encoding genes were synthesized, cloned into the pet32a (+) vector, and recombinant expression plasmids were constructed and transformed into BL21 (DE3). Recombinant protein (rNP) obtained from the optimized expression were purified over a Ni column and identified by SDS-PAGE and Western blot. The purified protein was used as the antigen to optimize the reaction conditions, and an indirect ELISA assay for GTV IgG antibody was developed and optimized, which was evaluated and initially applied.Results:The prokaryotic expression plasmid pet32a-NP was successfully constructed, the recombinant protein was highly expressed in E. coli in the form of inclusion bodies, the size was about 44 kD, and the results of Western blot indicated that the recombinant protein had good antigenicity with GTV positive serum. The optimized ELISA (GTV-rNP-iELISA) established in this study showed strong specificity, high sensitivity, and the coefficient of variation within and between batches is less than 10%, and has good repeatability; the detection results are consistent with the IFA detection results. Using the established ELISA method to detect 162 sheep sera from some regions of Xinjiang in 2017-2019, the total positive rate of antibodies was 39.8%.Conclusions:The GTV NP antibody detection ELISA method has good sensitivity, reproducibility, and specificity and has the potential to be a powerful tool for the diagnosis and serological investigation of GTV infection.
