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Published on: December 21, 2011
[Cloning and characterization of p43 cDNA from Trichinella spiralis]
Heng Guo1, Lian-rui Li, Ming-yuan Liu
1Institute of Zoonoses, College of Animal Science and Veterinary Medicine of Jilin University, Changchun 130062, China.
Objective:
To clone and characterize the p43 cDNA from muscle larvae cDNA library of Trichinella spiralis (Ts) Chinese isolate.
Methods:
PCR technique was used to amplify the target cDNA from muscle larvae cDNA library. After cloned in pMD18T vector, it was transformed into E. coli NovaBlue. The positive clones were sequenced and the cDNA was cloned into pET28a expression vector. After induced by IPTG, the inclusion body of the recombinant protein was purified and re-natured. The deoxyribonuclease II (DNase II) activity of the recombinant protein was tested by hydrolyzing XDNA.
Results:
Open reading frame (ORF) of the p43 cDNA was successfully cloned from the muscle larvae cDNA library of the Chinese Ts, there were mutations of two nucleotides in the ORF of the Chinese Ts p43 cDNA comparing with that from U.S.A. isolate at the positions 210 and 604, namely, C and A in the USA isolate but T and G in the Chinese isolate. Considering that three authors had cloned the same p43 cDNA from the USA isolate and six groups (including this team) had also obtained the same sequence from the Chinese isolate, the mutation of the two nucleotides was considered as the single nucleotide polymorphic (SNP) marker of the Chinese Ts isolate. The DNase II activity of the recombinant protein was successfully detected by hydrolyzing lamdaDNA.
Conclusion:
The p43 cDNA was successfully cloned from the muscle larvae cDNA library of the Ts Chinese isolate. Two SNPs were found in the nucleotide sequence. The DNase II activity was proved.
