[cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].
OBJECTIVE
To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system.


METHODS
The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG).


RESULTS
A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000.


CONCLUSIONS
The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.
Department of Respiratory Medicine, China Medical University, Shenyang, China.
Lei
L
Xu
Ling-ling
LL
Wang
Shuo
S
Liu
Yuan
Y
Ling
Lie
L
Ma
Qun
Q
Wang
Li-jiao
LJ
Zhang
Xiao-yu
XY
He
Ming-jing
MJ
Zhao
Xiao-ge
XG
Wang
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D013302:Streptomyces / Q000235:genetics* / Q000276:immunology
D000942:Antigens, Bacterial; D018076:DNA, Complementary