Expression of phosphoprotein P2 of Cysticercus cellulosae in Pichia pastoris and its application.
- Author:
Cai-Xia SU
1
;
Xue-Peng CAI
;
Xue-Qing HAN
;
Xue-Long LUO
;
Ya-Dong ZHENG
;
Yong-Xi DOU
Author Information
1. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Blotting, Western;
Cysticercosis;
diagnosis;
metabolism;
Electrophoresis, Polyacrylamide Gel;
Genome, Fungal;
genetics;
Helminth Proteins;
genetics;
metabolism;
Humans;
Phosphoproteins;
genetics;
metabolism;
Pichia;
Polymerase Chain Reaction;
Recombinant Proteins;
genetics;
metabolism;
Swine;
Taenia;
metabolism
- From:Chinese Journal of Biotechnology
2003;19(4):424-427
- CountryChina
- Language:Chinese
-
Abstract:
Cysticercosis is caused by the metacestode form of Taenia solium-Cysticercus cellulosae and it causes great economic losses and threatens the people's health. There are some problems on how to control cysticercosis, in order to resolve the key problem that the native antigen to diagnose and prevent cysticercosis is very limited and is not satisfied, Pichia pastoris Expression System was used to express recombinant P2 protein. The interested P2 gene was got by digesting the pGEM - P2 vector using restriction endonuclease, then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E. coli. Positive recombinant plasmids were selected sequenced and named pPIC9K-P2 and it was linearized by Sal I and Bgl II, then the linear DNA transfored into Pichia pastoris GS115 by electroporation. The recombinant expression vector pPIC9K - P2 integrated into GS115 via homologous recombination between the transforming DNA and regions of homology within the genome. The transformants were screened for multicopy recombinants using G418 and were distinguished for Mut phenotypes by MD and MM. Two different phenotypes were generated-HIS+ MUT+ (Methanol utilization plus) and HIS+ MUT(S) (Methanol utilization slow). PCR analysis of the multicopy recombinants indicated that the P2 gene was integrated within the genome of pichia Pastoris. The multicopy recombinants were named GS115/pPIC9K - P2HIS+ MUT+ and GS115/pPIC9K-P2HIS+ MUT(S), both HIS+ MUT+ and HIS+ MUT(S) were induced with methanol. The results of SDS-PAGE and Western blot demonstrated that the culture supernatant of the induced Pichia pastoris contained P2 protein which was accumulated up to 33 % of total proteins in the culture supernant and its molecular weight is 12.6kD. The results of the clinical study indicated that the expression P2 protein could be used to diagnose human cysticercosis and swine cysticercosis as diagnosis antigen.
