【Objective】To clone Cysticercus cellulosae AgB gene and express in vitro. 【Method】 The upper and downer fragments of AgB gene were amplified by RT-PCR, respectively. Then, the complete gene of AgB was cloned by splicing overlap extension PCR method using 35 nucleotides overlap between the upper and downer fragments and inserted into the vector pVAX1. This recombinant plasmid pVAX1/B was identified by digestion of endonuclease,PCR and sequencing.Then,it was transfected into BHK-21 cell line mediated by liposome 2000. Specific AgB protein expressed in BHK-21 cell line was detected by SDS-PAGE, Western-blotting and indirect immunofluorescence test. 【Result】 Electrophoresis analysis showed that the specific sequences were amplified and the sizes of products were accord with expectation, respectively. AgB was expressed in BHK-21 cells and was recognized by cysticercosis cellulosae positive serum. 【Conclusion】 Cysticercus cellulosae AgB gene has been cloned using splicing overlap extension PCR method, and expressed in BHK-21 cell line successfully.

Using different doses of recombinant porcine interferon-gamma(IFN-γ) to irritate abdominal macrophage and PK15 cell in vitro,effects of IFN-γ on the invasion and proliferation of Toxoplasma gondii were observed.The anti-T.gondii effect of macrophage was enhanced with the increase of dose of IFN-γ.Different concentrations of IFN-γ did not affect the T.gondii tachyzoites invading into PK15 cells.It is proposed that pIFN-γ may activate abdominal macrophage to inhibit the invasion and proliferation of T.gondii,and the anti-toxoplasma effect of macrophages activated by IFN-γ was dose-dependent,indicating that the role of IFN-γ on non-phagocytic cells was different from that on macrophage.

The cysteine protease TsCL-1 gene of Cysticercus cellulosae was amplified from the total RNA of C.cellulosae by RT-PCR.Then the fusion expression vector pPIC9K-TsCL-1 was constructed by inserting the amplified TsCL-1 gene into pPIC9K,and was transformed to GS115 by electroporation.Multicopy recombinant strains were screened by G418 and induced by methanol.SDS-PAGE was used to analyze expression product.The results showed that the gene shared 99.8% and 100% identity at the nucleotide and amino acid levels to the ones available in GenBank,and 76.3% amino acid similarity to cathepsin of Fasciola hepatica and Fasciola gigantica,respectively.The recombinant pTsCL-1 was expressed successfully in Pichia pastoris,and was approximately 42 ku in molecular mass.Western-blot analysis showed that the expressed recombinant protein had reactogenicity.

A pair of primers were designed according to the sequence of porcine IFN-γ mature protein gene.Then the amplified gene was cloned into pGEX-4T-1 vector and the fusion expression vector GST-pIFN-γ was constructed.The recombinant vector was transformed into Escherichia coli strain BL21.The recombinant strain was induced by IPTG and the induction conditions were optimized.Under the optimized conditions,the fusion protein GST-pIFN-γ was expressed.The soluble product was purified by GST aga-rose gel.The inclusion bodies were washed by DOC,and dissolved in SKL and subsequently renatured by dislysis.The purified recombinant protein was analyzed by SDS-PAGE and Western-blotting.The results showed that the purified fusion protein GST-pIFN-γ was obtained,and the recombinant protein was approximately 42 ku in molecular mass.The antiviral activity of the protein was determined by inhibiting the cytopathic effect,and the result showed that the fusion protein could suppress the cytopathic effect caused by FMDV(RNA virus) and PRV(DNA virus),and the anti-FMDV activity was higher than that of anti-PRV.In addition,the GST-pIFN-γ had the better stability and lower side effect.

Induction of formation of interferon-gamma production by specific inducting agent,as a kind of defense chemicals for maintains the stability of body generated by body self,is mainly secreted by NK cells and T cells.Due to the unique effect of anti-virus,anti-tumor and immune system,IFN-γ has a broad prospect of application in prevention and therapy of animal's infectious diseases.This article summarized porcine interferon-gamma briefly in following aspects,including production,structural characteristic,detection,biological activity,physiological mechanism and application.

Genomic DNA was extracted from the skin tissue of suspected capripox sheep in Jingtai, Gansu Province, and the glycoprotein-encoding orf118 gene was cloned by PCR. Comparison shows that the length of open reading frame of glycoprotein-encoding orf118 gene is 516 bp, coding for a 171-amino acid protein with molecular weight of approximate 19 500, and its nucleotide component showed a serious imbalance with 72.48% of A+T and 27.52%of G+C. The homology of nucleotide and deduced amino acid sequence between sheeppox virus ,Gansu strain and sheeppox virus TU-V02127 strain is 99.8% and 100%, respectively. orf118 of the sheeppox virus, Gansu strain shares 96.5% and 94.8% of identity with Lumpy skin disease virus and goatpox virus G20-LKV strain at a nucleotide level. Structural prediction of the deduced orf118 protein indicates that the orf118 antigen is a secretory glycoprotein with two N-linked glycosylation sites and one trans-membrane domain. Together with above results suggest that the orf118 glycoprotein is completely conserved among sheeppox field strains and has potential value for vaccine and diagnosis.