Construction of The Antigen Expression Vector Used for Library-library Screening
- VernacularTitle:用于库-库筛选体系的抗原表达载体的构建
- Author:
Rongkai GAO
;
Yan WANG
- Publication Type:Journal Article
- Keywords:
library-library screening, antigen, expression vector, selectively-infective phage (SIP)
- From:
Progress in Biochemistry and Biophysics
2006;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Selectively infective phage (SIP) technology was developed for screening interacting protein-protein pairs. The in vivo SIP strategy would in principle be suitable for??library-library??selections, and the co-packaged polyphage may be a suitable approach. In order to construct the antigen expression vector which can be co-packaged into polyphage with phage displaying vectors, plasmid TG10 was chosen as the basic vector which is compatible with antibody display vector. The interval sequence of phage genome was amplified with PCR and cloned into TG10 to provide the packaging signal. It was named pTMI and it can be packaged into phage particles in 1011 level. The N1N2 region of gene ￠? was amplified and cloned into pTMI under the control of lac promoter to give pTMIN. Promoter trc was synthesized and replaced the lac promoter to give pTTMIN which permits the fusion expression of antigen with N1N2. To test its ability for fusion expression, gene code for ten-peptide of c-myc was synthesized and inserted into pTTMIN downstream to N1N2. After induction expression, the results of ELISA and SDS-PAGE showed that it has been expressed successfully. When pTTMIN was transfected into cell carrying antibody display vector p3MHHB3, it was copackaged into phage particles in 0.3% to 55% after rescuing with helper phage VCSM13.From the results it can concluded that the antigen expression vector was constructed successfully and it can be used for library-library screening in theory.
