Construction of a recombinant-BCG containing the LMP2A and BZLF1 genes and its significance in the Epstein-Barr virus positive gastric carcinoma.
The signal peptide Ag85B of Bacillus Chalmette-Guerin (BCG) was used to construct a recombinant plasmid of BCG. The BCG-Ag85B gene and fused EBV LMP2A and BZLF1 genes were amplified and successively inserted into the Escherichia coli-BCG shuttle-vector pMV261. The recombinant plasmids were then amplified in E. coli DH5α and transformed into competent BCG. The expression of BZLF1 and LMP2A fusion proteins in recombinant-BCG (rBCG) was shown by Western blot. After the injection of recombinant-BCG into mice, antibodies against the fusion protein BZLF1 and LMP2A were measured by ELISA, and the cellular immune effects were determined by the lactate dehydrogenate (LDH) release assays. The results confirmed that the cloned genes of BCG-Ag85B and Z2A were correctly inserted into the vector pMV261. The recombinant plasmid pMVZ2A expressed Z2A in BCG effectively after transformation. The rBCG proteins were recognized by the BZLF1 (LMP2A) antibody. An ELISA demonstrated that rBCG could stimulate the generation of antibody against the fusion protein. The fusion gene was constructed successfully, and the rBCG induced humoral and cellular immune response in mice.
Department of Pathogenic Biology, Provincial Key Discipline of Medical Immunology, Jining Medical University, Shandong, China.
Qing-Jie
QJ
Xue
Jun
J
Dai
Xiu-Zhen
XZ
Li
Wei
W
Zhu
Chuan-Ping
CP
Si
Ting
T
Chen
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors; D008297:Male; D008810:Mice, Inbred C57BL; D009163:Mycobacterium bovis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D015534:Trans-Activators / Q000235:genetics / Q000276:immunology*; D014611:Vaccination / Q000379:methods; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology*; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; C063676:BZLF1 protein, Herpesvirus 4, Human; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014614:Vaccines, Synthetic; D014763:Viral Matrix Proteins; D014765:Viral Vaccines
BCG; EB virus; antibody; cellular immune; recombinant gene Z2A