[Expression, purification and activity assay of Streptococcus suis serotype 2 cell wall protein SSU1664].

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The recombinant SSU1664 protein has immunogen activity and might be one promising Streptococcus suis vaccine candidate and diagnosis marker of Streptococcus suis early infection.

[Purification and biological activities analysis of streptococcus suis Serotype 2 suilysin].

AIM: To explore the purification methods of wild-type and recombinant suilysin and to evaluate their biological activities. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Cloning, expression and preliminary crystallographic analysis of the equine infectious anaemia virus (EIAV) gp45 ectodomain.

Like human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV) belongs to the lentivirus genus. The first successful lentiviral vaccine was developed for EIAV. Thus, EIAV may serve as a valuable model for HIV vaccine research. EIAV glycoprotein 45 (gp45) plays a similar role to gp41 in HIV by mediating virus-host membrane fusion. The gp45 ectodomain was constructed according to the structure of HIV gp41, with removal of the disulfide-bond loop region. The protein was expressed in Escherichia coli and crystallized following purification. However, most of the crystals grew as aggregates and could not be used for data collection. By extensively screening hundreds of crystals, a 2.7 Šresolution data set was collected from a single crystal. The crystal belonged to space group P6(3), with unit-cell parameters a = b = 46.84, c = 101.61 Å, α = ß = 90, γ = 120°. Molecular replacement was performed using the coordinates of various lengths of HIV gp41 as search models. A long bent helix was identified and a well defined electron-density map around the long helix was obtained. This primary model provided the starting point for further refinement.

[Establishment of stable cell line and expression and purification of HIV gp120 in Drosophila S2 cells].

Constructing eukaryotic expressing vector of pMT/Bip/V5-His A-HIV gp120 and transfecting S2 cells to establish stable gp120-expressing S2 cell line. The gp120 gene of HIV CRF07-BC epidemic in China was amplified by polymerase chain reaction from the recombinant vector PRSV-gp120 and confirmed by DNA sequence analysis. The DNA fragment of HIV gp120 was digested with the restriction endonucleases NcoI and XhoI, then inserted into eukaryotic expressing vector pMT/BiP/V5-His A. A selection vector containing the streptomyces griseochromogenes bsd gene conferring blasticidin resistance was cotransfected into drosophila S2 cells by Cellfectin II reagent. The stable cell line was established following repeated screening by blasticidin. HIV gp120 was purified by a Ni-NTA affinity column followed by molecular sieve chromatography. Recombinant protein was characterized by SDS-PAGE, Western blot and ELISA. The eukaryotic expressing vector pMT/BiP/V5-His A-HIV gp120 was constructed, stable expressing cell line was established, and protein was expressed and purified successfully. This result will contribute to functional study of gp120 and vaccine design against AIDS.