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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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
Hongling
H
Department of Virology, School of Public Health, Shandong University, Jinan, People's Republic of China.
Wang
Zhiyu
Z
D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D012409:Rubella / Q000175:diagnosis*; D012412:Rubella virus / Q000276:immunology*; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C040901:rubella antibodies; C061826:E1 envelope protein, Rubella virus