[Gene synthesis, expression and immunogenicity analysis of TSP2 hydrophilic domain(TSP2HD) of Schistosoma japonicum].
OBJECTIVE
To synthesize and express the gene of TSP2 hydrophilic domain of Schistosoma japonicum, and investigate the immunogenicity of the recombinant TSP2HD protein.


METHODS
The whole DNA fragment encoding the TSP2 hydrophilic domain was synthesized by overlapping PCR, and confirmed by DNA sequencing. The recombinant plasmid TSP2HD-PG was constructed by inserting the purified TSP2HD DNA fragment into expression vector pGEX-4T-3 and the GST-TSP2HD fusion protein was expressed by transforming the recombinant plasmid TSP2HD-PG into Escherichia coli BL21 (DE3) and induced the recombinant with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The expressing situation of fusion protein was analyzed by SDS-PAGE. The GST-TSP2HD fusion protein was purified by affinity chromatography with glutathione sepharose 4B gel, and the purified recombinant TSP2HD protein was prepared by digesting the GST-TSP2HD fusion protein with thrombin. The immuno-response of the recombinant TSP2HD recognized by the pool sera of schistosomiasis patients and the pool sera of heavily infected rabbits was explored by Western blotting analysis. The immunogenicity of the recombinant TSP2HD was investigated by comparing the difference of counts per minute (cpm) value of lymphocyte proliferation test between experiment group and control group.


RESULTS
A 228 bp of TSP2HD gene fragment was obtained after overlapping PCR of three times and its DNA sequence was confirmed by DNA sequencing, which was same to one of the native TSP2HD. The recombinant containing recombinant plasmid TSP2HD-PG expressed a soluble fusion protein of GST-TSP2HD (Mr approximately 34 000) after being induced with IPTG. The purified recombinant TSP2HD protein was obtained through digesting the GST-TSP2HD fusion protein with thrombin. The recombinant TSP2HD was recognized by pool sera of schistosomiasis patients and pool sera of infected rabbits, indicating that the recombinant TSP2HD has a good response activity. The recombinant TSP2HD also stimulated proliferation of lymphocytes in infected mouse, the cpm value of experiment group was higher than that of the control (P < 0.01).


CONCLUSIONS
The Sj TSP2HD gene has been synthesized and expressed with immunogenicity which is similar to that of the native antigen.
Yu
Chuan-Xin
CX
Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China. chxnyu@163.com
Li
Jian
J
Yin
Xu-Ren
XR
Hua
Wan-Quan
WQ
Liang
You-Sheng
YS
Gao
Qi
Q
D000818:Animals; D003001:Cloning, Molecular; D018509:DNA, Helminth / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011817:Rabbits; D012549:Schistosoma japonicum / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D018509:DNA, Helminth; D015801:Helminth Proteins; D007106:Immune Sera; D008565:Membrane Proteins