[Cloning and characterization of p43 cDNA from Trichinella spiralis].
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.


CONCLUSIONS
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.
Institute of Zoonoses, College of Animal Science and Veterinary Medicine of Jilin University, Changchun 130062, China.
Heng
H
Guo
Lian-rui
LR
Li
Ming-yuan
MY
Liu
Xiu-ping
XP
Wu
Shu-min
SM
Sun
Bao-quan
BQ
Fu
Chang-ling
CL
Gao
Qiang
Q
Lu
Qi-jun
QJ
Chen
Pascal
P
Boireau
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D015723:Gene Library; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*; D017160:Trichinella spiralis / Q000235:genetics*
D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C087692:SAG3 protein, Toxoplasma gondii; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II