ABSTRACT: To construct the suilysin mutant without hemolytic activity and evaluate its functions.
The proline in 353 site of suilysin was site-directed mutated to alanine, leucine and valine, respectively. The recombinant mutants were renaturated and purified by immobilized metal ion affinity chromatography, and the purified proteins were evaluated in the hemolytic activity and immunogenicity.
We obtained three mutants, SLY(P353A), SLY(P353L) and SLY(P353V). The SLY(P353V) mutant had non-hemolytic activity. Western blotting and animal experiments showed that SLY(P353V) mutant still had immunogenicity.
Suilysin mutant SLY(P353V) has no hemolytic activity but remains immunogenicity.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 06/2012; 28(6):580-2.
ABSTRACT: To amplify the SSU1664 gene from Streptococcus suis serotype 2 strain 05ZY, to express the gene in E.coli, and to evaluate the activities of the recombinant protein.
SSU1664 gene was amplified by PCR using primers according to 05ZY genome sequences and cloned into the expression vector. The recombinant protein was purified by affinity chromatography and its immunogen activities were tested by Western blot and ELISA.
SSU1664 gene could solublely express in E.coli BL21(DE3). Western blot analysis showed that the recombinant protein could react with rat serum immunized with Streptococcus suis, but not with non-immunized rat serum. ELISA assay showed that anti-SSU1664 IgM content in Streptococcus suis-infected patient was significantly higher than that in healthy donors.
The recombinant SSU1664 protein has immunogen activity and might be one promising Streptococcus suis vaccine candidate and diagnosis marker of Streptococcus suis early infection.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 07/2011; 27(7):757-9.
ABSTRACT: To explore the purification methods of wild-type and recombinant suilysin and to evaluate their biological activities.
Wild-type suilysin was purified by ammonium sulfate precipitation, anion-exchange chromatography and hydrophobic chromatography in turn, while recombinant suilysin was first refolded and purified by immobilized metal ion affinity chromatography, and further purified by Thiopropyl Sepharose 6B. The biological activities were evaluated by hemolysis test, cytotoxicity assay.
Both prepared wild-type and recombinant suilysin, with purify over 90%, have hemolysis activity and could injure target cells at high concentration while cholesterol could completely inhibit their activities.
Recombinant suilysin has similar biological activities with wild-type suilysin, and this work contributed to further study the functions of suilysin on pathogenesis of steptococcus suis.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2011; 27(4):374-6.