Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases · 2010
[Gene synthesis, expression and characterization of egg protein IPSE of Schistosoma mansoni].
Аннотация на английскомСтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод пока не подготовлен
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Xie Shu-ying, Chen Hong-gen, Yu Chuan-xin, Yin Xu-ren, Hua Wan-quan, Zeng Xiao-jun, Liang You-sheng, Gao Qi
Аннотация
OBJECTIVE: To synthesize and express the gene of egg protein IPSE (IL-4-inducing principle of Schistosoma mansoni eggs) and evaluate its immunologic characteristics.
METHODS: The IPSE gene of S. mansoni was synthesized by overlapping PCR, and confirmed by DNA sequencing, The recombinant plasmid IPSE-pET32a(+) was constructed by inserting the gene of IPSE into expression vector pET32a(+) at the downstream of thioredoxin (Trx) coding sequence. The recombinant plasmid IPSE-pET32a(+) was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. Large-scale fusion protein was prepared and purified with Ni affinity chromatography gel under denaturing conditions. A small amount of soluble Trx-IPSE was obtained by dialyzing the fusion protein in a large volume of PBS. Western blotting was used to detect if the recombinant IPSE was recognized by the IgG antibody in the pooled patient sera of schistosomiasis japonica and its binding capacity to the non-specific IgE antibody in the sera of healthy persons.
RESULTS: DNA sequencing confirmed that the nucleotide sequence of synthesized IPSE gene was completely identical to the native one. SDS-PAGE showed that the recombinant plasmid IPSE/pET32a(+) expressed a fusion protein with an Mr 35700 after being induced by IPTG. The pure fusion protein Trx-IPSE reacted positively with the pooled sera of schistosomiasis patients under either denaturing or renaturing conditions. The protein Trx-IPSE also reacted with the nonspecific IgE in the sera of healthy persons.
CONCLUSION: The IPSE gene of Schistosoma mansoni has been synthesized, and the recombinant can react with natural antibody IgG against S. japonicum and non-specifically bind to IgE antibody.
METHODS: The IPSE gene of S. mansoni was synthesized by overlapping PCR, and confirmed by DNA sequencing, The recombinant plasmid IPSE-pET32a(+) was constructed by inserting the gene of IPSE into expression vector pET32a(+) at the downstream of thioredoxin (Trx) coding sequence. The recombinant plasmid IPSE-pET32a(+) was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. Large-scale fusion protein was prepared and purified with Ni affinity chromatography gel under denaturing conditions. A small amount of soluble Trx-IPSE was obtained by dialyzing the fusion protein in a large volume of PBS. Western blotting was used to detect if the recombinant IPSE was recognized by the IgG antibody in the pooled patient sera of schistosomiasis japonica and its binding capacity to the non-specific IgE antibody in the sera of healthy persons.
RESULTS: DNA sequencing confirmed that the nucleotide sequence of synthesized IPSE gene was completely identical to the native one. SDS-PAGE showed that the recombinant plasmid IPSE/pET32a(+) expressed a fusion protein with an Mr 35700 after being induced by IPTG. The pure fusion protein Trx-IPSE reacted positively with the pooled sera of schistosomiasis patients under either denaturing or renaturing conditions. The protein Trx-IPSE also reacted with the nonspecific IgE in the sera of healthy persons.
CONCLUSION: The IPSE gene of Schistosoma mansoni has been synthesized, and the recombinant can react with natural antibody IgG against S. japonicum and non-specifically bind to IgE antibody.