[Expression of HPV16 E7 protein and preparation of its polyclonal antibody].
OBJECTIVE
To prepare a prokaryotic expression vector carrying E7 protein of human papillomavirus (HPV) type 16 and a polyclonal antibody against it.


METHODS
The HPV16 E7 gene was amplified by PCR from the tissue samples of cervical cancer and cloned into the pET21a(+) prokaryotic expression vector. The constructed pET21a(+)/HPV16 E7 recombinant plasmid was transformed into E.coli BL21(DE3) and induced by isopropyl beta-D-1-thiogalactopyranoside (IPTG). The recombinant protein of HPV16 E7 was purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis. To prepare the polyclonal antibody, the rabbits were immunized with the purified recombinant protein HPV16 E7. The titers of specific antibodies were detected by ELISA. The specificity activity was further detected by Western blotting and immunofluorescence analysis.


RESULTS
HPV16 E7 recombinant protein was expressed and purified in the prokaryotic expression system. The polyclonal antibodies were produced in the rabbits immunized with the recombinant protein. The titer of specific antibodies was up to 1:30 000. Western blotting and immunofluorescence analysis indicated that the polyclonal antibody could specifically recognize the HPV16 E7 protein.


CONCLUSIONS
HPV16 E7 recombinant protein was successfully expressed and its polyclonal antibody was prepared.
Institute of Molecular Virology and Immunology, Department of Medical Microbiology and Immunology, Wenzhou Medical University, Wenzhou 325000, China.
Bingbing
B
Wang
Jianxin
J
Tu
Yan
Y
Lv
Bolong
B
Hou
Qiaoqiong
Q
Liu
Xiaoyun
X
Lin
Shao
S
Chen
Xiangyang
X
Xue
Shanli
S
Zhu
Lifang
L
Zhang
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007114:Immunization; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D050725:Papillomavirus E7 Proteins / Q000235:genetics* / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits
D000906:Antibodies; D007074:Immunoglobulin G; D050725:Papillomavirus E7 Proteins; C059731:oncogene protein E7, Human papillomavirus type 16