Expression and characterization of rubella virus glycoprotein E1 in yeast cells.

To express E1 glycoprotein of rubella virus (RuV) strain JR23 in yeast and develop a diagnostic assay using expressed E1 protein as coating antigen in comparison with other diagnostic assays. cDNA of E1 open reading frame of RuV was PCR amplified using plasmid pMD18-T-E1 as template and cloned into plasmid pBluscriptII SK+. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pBluscriptII SK(+)-E1 plasmid DNA was digested by restriction endonuclease EcoR I and Xba I, and a fragment of 1.5 kb was isolated and cloned into a yeast expression pGAPZ(alpha)A, resulting in pGAPZ(alpha)A-E1. After confirmation by sequencing, pGAPZ(alpha)A- E1 was transformed into yeast GS115 cells with LiCl method. E1 protein expression in GS115 was analyzed by SDS-PAGE and Western blot. An indirect ELISA was developed using the recombinant E1 protein as coating antigen for detecting RuV E1 antibodies in 90 serum samples. To compare the specificity, sensitivity and reproducibility of the assay with other methods, the same serum samples were also assayed by RuV culture medium as coating antigen (Jingmei kit and German RECI kit). Statistical analyses were performed to compare the differences among these methods and to determine which coating antigen source, the recombinant E1 protein or RuV-infected culture medium, is more suitable for the assay. A fragment of 1.5 kb, corresponding to the full open reading frame of E1, was PCR amplified and cloned in yeast expression vector. The clone was confirmed by restriction digestion, PCR and sequencing. E1 as a secretive protein was successfully expressed by GS115. Its molecular weight was about 58 kDa. SDS-PAGE showed that the recombinant protein was expressed efficiently and constantly in Pichia pastoris GS115 cells. The expression level reached a peak 48 h after culturing and stabilized thereafter. E1 protein was detected in both supernatant and cells. Western blot showed that the secretive E1 protein in the supernatants could react with both the anti-RuV-positive serum and a monoclonal antibody against E1. However, E1 protein derived from cells could only react with the anti-RuV-positive serum, polyclonal antibody, but not the monoclonal antibody. Compared with the German RECI kit, the sensitivity, specificity, and accordance rate of the assay using recombinant E1 protein as coating antigen were 67.11, 71.43 and 67.78%, respectively, while those of the assay using RuV-infected culture medium as coating antigen were 50, 78.57 and 54.44%, respectively. Compared with the German RECI kit, the sensitivity, specificity, and accordance rate of the ELISA assay using the Jingmei kit were 84.71, 71.43 and 82.22%, respectively. The data indicated that recombinant E1 protein derived from the yeast expression system can serve as a better source than RuV-infected cell medium as coating antigen for detecting antibodies against RuV in the indirect ELISA assay. Statistical analysis of the data generated from two independent experiments using recombinant E1 protein as coating antigen indicated that the assay was very consistent with no statistically significant difference between the two experiments (p > 0.05). 76 out of 90 serum samples were detected positive using the German RECI kit, while 68, 55 and 41 samples were positive using the Jingmei kit, recombinant E1 and RuV-infected cell medium, respectively. Statistical analyses indicated that the positive rates were significantly different among all four assays (p < 0.05) except for one pair (German RECI kit and the Jingmei kit: p > 0.05). Comparing the positive rates obtained from the assay using recombinant E1 and that using RuV-infected cell medium, it seems that the recombinant E1 protein is a better source than RuV culture medium as coating antigen in the indirect ELISA assay for detection of RuV antibody. The recombinant yeast expression vector of RuV E1 glycoprotein was constructed successfully. The E1 protein as a secretive protein was successfully expressed by GS115 and maintained its antigenicity very well. As coating antigen, the recombinant E1 protein served a better source than RuV culture medium in the indirect ELISA method for the detection of RuV antibody.

Wen H ，Wang Z 《INTERVIROLOGY》

[Expression and bioassay of rubella virus E1-374 glycoprotein in yeast cells].

Li ZM ，Wen HL ，Lin B ，Sun CX ，Chu FL ，Yuan XJ ，Song YY ，Xu HZ ，Wang ZY ... -  《-》

Expression of E2 gene of bovine viral diarrhea virus in Pichia pastoris: a candidate antigen for indirect Dot ELISA.

The E2 gene containing the EcoR I and Not I sites of bovine viral diarrhea virus (BVDV) was amplified from the plasmid pMD-18T-E2 of the HB-bd isolated, and inserted into Pichia pastoris (P. pastoris) expression vector pPIC9K, and transfected into Escherichia coli DH5α. The recombinant plasmid pPIC9K-E2 was digested by the SalI restriction enzyme and transformed into the P. pastoris strain GS115 by electroporation. High copy integrative transformants were obtained by G418 screening and induced for expression with methanol. The expressed products in the culture medium were identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the Western blotting and the antibody test for immunity. An indirect Dot-ELISA for the detection of antibody against BVDV was established by the recombinant E2 protein as the coating antigen. The reaction conditions of the indirect Dot-ELISA were optimized. The coating concentration of the E2 recombinant protein antigen, the dilution of serum sample, the optimal concentration of HRP labeled antibody, the optimal blocking reagent and blocking time were studied. 100 sera samples from cows in the field were tested for the antibody against BVDV by the Dot-ELISA and the IDEXX HerdChek BVDV antibody ELISA kit simultaneously to compare the specificity, sensitivity and accuracy. The results showed that the expressed products in the culture medium resulted in single band of 44kDa by SDS-PAGE and Western blotting. The results of the immunogenicity assay indicated that the protein E2 expressed in P. pastoris could induce the experimental animals of the rabbit to produce BVDV specific antibodies. The results of the indirect Dot-ELISA showed that the optimal coating concentration of the E2 recombinant protein was 2.0μg/mL, the bovine serum dilution was 1:100, the optimal concentration of HRP-labeled rabbit anti-bovine antibody IgG was 1:500, and the optimal blocking reagent was 3% glutin-TBS and blocking for 45min. The indirect Dot-ELISA showed 96.7%, 92.5% and 95% in the terms of specificity, sensitivity and accuracy compared to the IDEXX ELISA test kit. The indirect Dot-ELISA using the E2 recombinant protein can be used for the detection of antibody against the BVDV and could be considered in the surveillance programs.

Zhao Y ，Ma T ，Ju X ，Zhang Y ，Wang M ，Liu T ，Cao W ，Bao Y ，Qin J ... -  《-》

Development of an indirect ELISA for serological detection of reticuloendotheliosis virus using the gp90 protein expressed in Pichia pastoris.

The present study was undertaken to express the gp90 protein of reticuloendotheliosis virus (REV) in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The full-length gp90 gene of REV was cloned into pPIC9k vector and then integrated into the chromosome of P. pastoris for induced expression. SDS-PAGE and western blot assay demonstrated that gp90 protein was expressed and secreted into the culture medium at about 100mg/L of culture under optimized condition. An indirect ELISA was then established by using the recombinant gp90 protein as the coating antigen. The optimal concentration of coated antigen was 0.1 μg/well at a serum dilution of 1:200 and the optimal positive threshold value of the assay was 0.409. Cross-reactivity assay showed that this antigen was REV specific. The reproducibility experiment displayed good consistency. Furthermore, the gp90 protein based indirect ELISA showed good correlation rates of 96.3% and 97.5% with virus neutralization test and a commercially whole virus based indirect ELISA, respectively. This study demonstrates the efficacy of recombinant gp90 protein as an antigen in ELISA for seroepidemiological study of REV infection on a large scale.

Li K ，Gao H ，Gao L ，Qi X ，Gao Y ，Qin L ，Wang Y ，Wang X ... -  《-》

Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA.

Bhanot V ，Balamurugan V ，Bhanuprakash V ，Venkatesan G ，Sen A ，Yadav V ，Yogisharadhya R ，Singh RK ... -  《-》