Related Experiment Videos
[Construction and screening of hepatitis E virus-specific phage antibody combinatorial library]
Hua Wei1, Jian-Qiong Zhang, Hai-Qin Lu
1Genetic Research Center, Medical School, Southeast University, Nanjing 210009, China. wei.xie@seu.edu.cn
Aim:
To construct HEV-specific phage combinatorial anti-body library and screen anti-HEV antibodies with neutralizing activity from the library.
Methods:
The total RNA was extracted from B-lymphocytes of 6 HE patients. Kappa chain and Fd segment of IgG gene were amplified respectively by RT-PCR using a set of Fab-specific primers. The amplified gene were inserted successively into vector pComb3 and electrotransformed E. coli XLI-Blue cells. Furthermore, the recombinant phage was rescued by being concultured with helper phage VCSM13 to construct HEV-specific phage anti-body library.
Results:
Fab displayed on the surface a as fusion protein with the N terminal of coat protein III, and 1. 8 x 10(7) clone library was established. Specific antibodies to HEV ORF2 recombinant antigen were acquired after five rounds of panning with HEV ORF2 recombinant antigen including neutralizing epitope.
Conclusion:
Four clones exhibited specific binding to HEV ORF2 recombinant antigen including neutralizing epitope is identified by ELISA. The results show that we have got the recombinant phage antibodies.