Construction and expression of human anti-HBs-IFN fusion gene
- VernacularTitle:人源抗HBsAg dsFv抗体靶向干扰素重组质粒的构建与表达
- Author:
Le JIANG
;
Jinqi YAN
;
Bingran GUO
;
Jie REN
;
Jiyun YU
- Publication Type:Journal Article
- Keywords:
interferon-alpha;
hepatitis B surface antigens;
disulfide stabilized Fv fragments;
plasmids;
gene expression
- From:
Medical Journal of Chinese People's Liberation Army
1981;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct eukaryotic expression plasmid pEE14.1-dsFv?pr+,and detect the expression of the recombined gene in eukaryotic CHO-K1 cells.Methods The cationic DNA fragment was cloned into the 3' of VH gene by overlapping extension PCR,and the 6?His tab was inserted to the 3' of VL and human IFN-? gene by the same way.The above mentioned recombinant VH and VL genes were inserted into a pCI-GPI vector first,and then cloned into the pEE14.1 vector to construct the recombinant plasmid pEE14.1-dsFv?pr+.Finally,the recombinant plasmid was transfected into the CHO-K1 cells by LipofectamineTM 2000,and the expression was detected by RT-PCR,ELISA and Western blotting.Results The enzyme digestion and sequencing analysis showed that the recombinant plasmid was successfully constructed.RT-PCR showed that only the cells with transfected plasmid can generate the specific 1700bp fragment.ELISA analysis showed that the production of IFN-?expressed in the supernatant of transfected cells was about 1.1ng/ml.Also,Western blotting could reveal the characteristic band of HBsAg dsFv?pr+ protein.Conclusion The antibody targeting to human IFN-?genes has been successfully expressed in a single open reading frame.Changing the electricity of the antibody may provide the necessary condition for the study of the a new type of anti-HBV drug in nanoscale in the future.
