[Construction of phage display cDNA library from adult worms of Schistosoma japonicum].
OBJECTIVE
To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum.


METHODS
Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library.


RESULTS
Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library.


CONCLUSIONS
The T7 phage display library from adult worms of Schistosoma japonicum was constructed.
Sun
Yi
Y
Shanghai Veterinary Research Institute of CAAS, National Laboratory of Animal Schistosomiasis Control, Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai 200232, China.
Jia
Ren-chu
RC
Liu
Jin-ming
JM
Yuan
Chun-xiu
CX
Shi
Yao-jun
YJ
Lu
Ke
K
Fu
Zhi-qiang
ZQ
Sun
Huan
H
Cai
You-min
YM
Lin
Jiao-jiao
JJ
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D016133:Polymerase Chain Reaction; D012549:Schistosoma japonicum / Q000235:genetics*
D018076:DNA, Complementary; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII