[Construction and prokaryotic expression of recombinant gene EGFRvIII HBcAg and immunogenicity analysis of the fusion protein].
OBJECTIVE
To construct recombinant prokaryotic expression plasmid pET28a(+)/c-PEP-3-c and evaluate the immunogenicity of the fusion protein.


METHODS
cDNA fragment encoding PEP-3 was obtained from pGEM-T Easy/PEP-3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni(2+)-NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA.


RESULTS
The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1:16 000 after the fourth immunization and reached 1:2.56x10(5) after the sixth immunization. The titre of anti-PEP-3 antibody reached 1:1.28x10(5) and the titre of anti-HBcAg antibody was less than 1:4x10(3).


CONCLUSIONS
Fusion gene PEP-3-HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII.
Duan
Xiao-yi
XY
Department of Nuclear Medicine, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China. duanxy@mail.xjtu.edu.cn
Wang
Jian-sheng
JS
Guo
You-min
YM
Han
Jun-li
JL
Wang
Quan-ying
QY
Yang
Guang-xiao
GX
D000818:Animals; D000918:Antibody Specificity; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000235:genetics*; D006512:Hepatitis B Core Antigens / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology*
D018076:DNA, Complementary; D006512:Hepatitis B Core Antigens; D011993:Recombinant Fusion Proteins; C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors