[Construction and immunoscreening of cDNA library of Babesia orientalis].
OBJECTIVE
To construct a cDNA library for Babesia orientalis and screen immunologically positive clones.


METHODS
Total RNA of B. orientalis in red blood cells from an infected calf was isolated. cDNA was synthesized by reverse transcriptase, amplified by PCR and ligated into lambdaTriplEx2 vector. The recombined vectors were packaged and the unamplified cDNA library was constructed. The cDNA library was then amplified and immunologically screened with rabbit anti-B. orientalis serum. The recombinant lambdaTriplEx2 of positive clones were converted to the corresponding recombinant pTriplEx2. The inserted fragments were identified by PCR amplification. The plasmids were sequenced and compared against GenBank database by Blast.


RESULTS
The titer of the unamplified library was 2.0 x 10(6) pfu/ml. The inserted fragment length of the library ranged from 500 to 3,000 bp, and the recombination efficiency accounted for 98.8%. The titer of the amplified library was 5.8 x 10(8) pfu/ml. Three positive clones were selected by serum immunological screening and named B04, B05, and B41, respectively. The inserted fragments of the B04, B05 and B41 were about 1,300 bp, 1,000 bp, and 2,400 bp, respectively. Sequence analysis revealed that the 3 clones contained open reading frames. Blast results showed that they were highly homologous to the nuclear movement protein gene, the hypothetical protein gene and the heat shock protein 70 (HSP70) gene, respectively. The deduced amino acid sequences of B04, B05 and B41 contained 310, 192 and 647 amino acid residues, with Mr of 34,000, 21,000, and 70,700, respectively.


CONCLUSIONS
A qualified cDNA library of B. orientalis has been constructed and three positive clones of B. orientalis discovered.
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Centre for Malaria, Schistosomriasis and Filariasis, Shanghai 200025, China.
Qin
Q
Liu
Dan-Na
DN
Zhou
Yan-Qin
YQ
Zhou
Ying
Y
Zhang
Lan
L
He
Bao-An
BA
Yao
Jun-Long
JL
Zhao
D000595:Amino Acid Sequence; D000818:Animals; D016793:Babesia bovis / Q000235:genetics* / Q000276:immunology*; D002020:Buffaloes / Q000469:parasitology; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D008297:Male; D011817:Rabbits; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary