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Updated: Apr 26, 2026
Functional Cloning Using a Xenopus Oocyte Expression System
Published on: January 30, 2016
[Cloning, expression and transcription specificity analysis of two tyrosinases from Schistosoma japonicum]
Objective:
To clone and express the recombinant proteins based on the whole open reading frame of two tyrosinases (tyrosinase 1 and tyrosinase 2) from Schistosoma japonicum, and study the transcription specificity of the two tyrosinases in different sex and developmental stages of S. japonicum.
Methods:
The full-length of SjTYR1 and SjTYR2 were amplified with specific primers and subcloned into pSJ2. The recombinant plasmids were transformed into E. coli Rosetta Gami strains and induced with IPTG for expression. The recombinant proteins were purified by Ni-NTA agarose. The recombinant proteins SjTYR1 and SjTYR2 were used to produce the specific antibodies by immunizing the rabbits. The immunogenicity of the recombinant proteins SjTYR1 and SjTYR2 were detected by Western blotting using sera of recombinant proteins-immunized rabbits and S. japonicum-infected rabbit serum as the primary antibody, respectively. The reactivity of sera from recombinant proteins-immunized rabbits was analyzed by Western blotting against the native protein of S. japonicum worm. Total RNA was extracted from 14, 16, 18, 20, 22, 24, 26, and 28-day male and female worms. Transcription levels of the two tyrosinases in different sex and different stage were determined via RT-PCR method.
Results:
The expression vector of SjTYR1/pSJ2 and SjTYR2/pSJ2 were constructed and the recombinant pro teins SjTYR1 and SjTYR2 were expressed in inclusion body in E. coli (about M(r) 55 000 and M(r) 56 800). The sera of S. japonicum-infected rabbits reacted positively with the purified recombinant protein SjTYR1, but not with recombinant protein SjTYR2. The native protein of S. japonicum worm could be recognized by sera of rSjTYR1-immunized rabbits (M(r) 100 000), but not by sera of rSjTYR2-immunized rabbits. Transcription levels of the two tyrosinases in male worms were nearly zero. In female worms, the transcription levels of the two tyrosinases increased sharply from the 24th day post-in- fection and reached maximum on the 28th day.
Conclusion:
The recombinant proteins of SjTYR1 and SjTYR2 show immunogenicity and immunoreactivity. SjTYR1 and SjTYR2 are both expressed specifically in female worms and the transcription levels increase in 24-28 days after infection.
