Expression of VCA genes of Epstein-Barr virus in Pichia pastoris and clinical application / 中华微生物学和免疫学杂志

ABSTRACT

Objective To analyze the application of recombinant VCA-BALF4(S) and VCA-BFRF3 proteins in serological diagnosis of nasopharyngeal carcinoma (NPC). Methods DNA extracted from the B95-8 cells was used as the templates in a polymerase chain reaction(PCR). VCA-BALF4 1008 bp (S) (aa 287-623) and VCA-BFRF3 531 bp(177 as) were generated and inserted into pPICZaA vector.The recombinant plasmids were transformed into GS115 yeast by electroporation. The yeast transformants in-duced by methanol expressed recombinant proteins. The recombinant proteins synthesized were coated to mi-croplate for detection of EBV-IgA antibody in NPC patients by ELISA. Results We have successfully se-cretly expressed the recombinant VCA-BALF4(S) and VCA-BFRF3 protein in the Pichia pastoris. The mo-lecular weight of products were approximately 37×10~3 and 18×10~3, respectively. The recombinant proteins VCA-BALF4(S) and VCA-BFRF3 in the culture supematant showed good immunoreactivity with IgA anti-bodies to EBV by Western blot. A novel ELISA was established using Pichia pastoris-expressed VCA-BALF4 (S) and VCA-BFRF3 proteins. Serum samples were collected from patients with NPC and healthy controls and using this ELISA tested. The sensitivity of VCA-BALF4(S) and VCA-BFRF3 tests in the NPC sera were 88.7% and 71.0%, whereas the specificity of normal individuals are 96.4% and 91.8% separately. Con-clusion The recombinant proteins BALF4 (S) and BFRF3 were highly secretly expressed in Pichia pastoris,the diagnostic value of two recombinant proteins in screening for NPC patients were primary evaluated and the valuable pPICZa-BALF4(S) yeast strain was obtained.