Most diseases are complex genetic traits caused by multiple genetic and environmental components. It has been proposed that common genetic variations, mainly single nucleotide polymorphisms (SNPs), influence the susceptibility to complex diseases. We have conducted an extensive review on the characters of SNPs, the related website information, and the genotyping methods of SNPs such as direct sequencing, SnaPshot, Taqman, real time quantitative (kinetic) PCR with allele specific amplification, denaturing high performance liquid chromatography, and OLA/PCR.The strategies for studying the relation between SNPs and complex diseases susceptible genes were also reviewed.

Objective: To investigate the structural characteristics and the primary functions of antigen gene cC1. Meth-ods: The primary and the secondary structures were analyzed using bioinformatics programs provided by Internet servers.The predicted 3D structure of cC1 was established by homology protein modeling method. Anticoagulant activity of GST-anx32 was assayed by modified kaolin partial thromboplastin time (KPTT). Results: cC1 had high homology to annexinsgenes both at nucleic acid and at amino acid level. It contained 4 homologous regions, and each region included the typical an-nexin motif "G-X-G-T (38 residues)-D/E". The results of KPTT assay showed that the recombinant protein GST-anx32 hadhigh anticoagulant activity. Conclusion: cC1 has the common structures of annexins but the homology to the extant annexinsis no more than 48%, cC1 is a member of a novel annexin subfamily and designed as annexin32.

AIM:To understand the interaction between a195- kilodalton protein,P195, on the surface of Plasmodium falciparum merozoite and human erythrocyte.METHODS:P195 was expressed in eight fragments in E.coli.After being refolded,the expressed proteins were la- belled with12 5 I,and incubated with human erythrocytes.RESULTS:According to binding assay, three fragments of P195:M3,M6,M9were found to have ability to bind to human erythrocyte. M6,which is equal to amino acid( AA) sequence from384 to595,could bind to human erythro- cytes but not to trypsin treated human erythrocytes,and the binding could be eluted by low p H buffer solution. M3( AA 12 3to 30 2 ) and M9( AA 10 78to 12 51) also have the ability to bind to human erythrocytes,but the binding was not affected by trypsin treatment and low p H buffer elu- tion. CONCL USION:The binding site of M6might be a surface protein receptor of human ery- throcytes,while the binding site of M3and M9might be an intracellular componentof human ery- throcyte.

Objective: To construct an eukaryotic expression plasmid containing gene coding for the hemagglutinin neuraminidase(HN) of Newcastle disease virus (NDV), and to study its mechanism and value in antitumor therapy. Methods: The HN cDNA was abtained from NDV with RT PCR and an eukaryotic expression vector of HN gene ( pcDNA3 HN ) was constructed. The antitumor effect was evaluated after injecting pcDNA3 HN into mice bearing B16 melanoma. Results: The HN cDNA of NDV was successfully cloned and pcDNA3 HN had a good expression in COS 7 cells. Animal experiments suggested that the pcDNA3 HN could significantly increase CTL and NK activity of tumor bearing mice. Conclusion:The eukaryotic expression plasmid containing the gene coding for the (HN) has the function of increasing CTL and NK activity of tumor bearing mice.

Objective:To investigate the immume mechanism and protective effect of DNA vaccine pcD flaB against pathogenic leptospira infection. Methods: DNA vaccine pcD flaB was constructed by inserting flaB into the eucaryotic expressing plasmid pcDNA3. After guinea pigs were infected with leptospira, the protective rate was observed and specific anti leptospira antibody IgG were tested by ELISA. TNF activity was tested by cell proliferation. Results:The protective rate against leptospira infection was 100%. The specific antibody IgG generated peaked at the 6th week. Activity of TNF released by macrophage of guinea pigs given DNA vaccine was higher than that not given vaccine. Conclusion:DNA vaccine pcD flaB can protect guinea pigs from leptospira challenge infection by inducing humoral immune response and increasing TNF activity.

Objective: To fuse Mycobacterium tuberculosis protective antigen gene with mice ubiquitin gene, constructing antigen ubiquitin system. Methods: Mice ubiquitin cDNA was amplified by RT PCR from mice testicle,and 4 antigen genes were obtained by PCR from cultured Mycobacterium tuberculosis . Ubiquitin and 4 antigen genes were linked by flexible adaptor respectively and the fusing genes were cloned into pVAX vector.The recombinant plasmids were digested with endonuclease and sequenced.Then the recombinant plasmids were transfected into COS7 cells and the expression was assayed by ELISA. Results:Ubiquitin and 4 antigen genes were 0.2,0.3,0.7,1.0,1.65 kb in length by agarose electrophoresis. Endonuclease digestion of the recombinant plasmids indicated that the fusion genes were correctly inserted into pVAX vector. Sequencing results of fusion nucleic acid vaccines were identical to those in GenBank.The recombinant plasmids expressed in COS7 cells. Conclusion: Four Chinese Mycobacterium tuberculosis protective antigens ubiquitin systems are successfully constructed and can be expressed in eukaryotic cells. This may provide a basis for diagnosis and therapy of tuberculosis.

The ribosomal DNA fragment was amplified with PCR utilizing synthetic primers with host bacteria chromosomal DNA as template. The resultant probes were labeled with Digoxigenin and were applied in the dot blot of DNA vaccine samples. The host bacteria genomic DNA in DNA vaccine against hepatitis B was less than 15 ng/?g. Digoxigenin labeled probes proved sensitive and reliable in determining genomic DNA.

Objective: To clone porcine IL-4 cDNA and observe its adjuvanticity of vaccine against T. solium cysticerco-sis. Methods:The cDNA of porcine IL-4 was amplified by RT-PCR, which had 5' AUG initiatory codon with optimized trans-lational initiation. After sequencing,the cDNA was intergrated into expression vector pcDNA and transient expression was performed. Then newborn pigs were immunized with IL-4 expression vector and protective antigen DNA vaccine against T. solium cysticercosis. Results:The obtained sequence of porcine IL-4 cDNA was the same as reported. IL-4 and protective antigen could effectively promote the humoral response. Conclusion:An efficient expression plasmid containing porcine IL-4 cDNA is constructed. Its adjuvanticity of DNA vaccine against T. solium cysticercosis is preliminarily conformed.

Objective:To identify the HLA-A0201 restricted CTL epitopes derived from hepatitis B virus X protein predicted by computer program and general principles in vitro.Methods:HBx gene sequences of Hepatitis B virus genotypes B/C and serotypes adw/adr,with the highest frequencies in Chinese,were computed and analyzed by screening service offered by Internet combined with peptide supermotif,extended motif and quantitative motif prediction.Four most ideal nine-peptides(HBx1,HBx2,HBx3,and HBx4)were selected as candidate peptides.Using flow cytometry,the fluorescence index of both control and experimental groups were detected and the 4 nine-peptides were evaluated with T2 binding assay and DC50 assay.Results:The nine-peptides VLCLRPVGA(HBx1),CLFKDWEEL(HBx2),VLHKRTLGL(HBx3)and HLSLRGLPV(HBx4)were selected as candidate targets.Among the 4 candidate peptides,HBx2 showed higher HLA-A0201 affinity and HBx2,HBx4 showed better stability.Conclusion:Our study indicates that CLFKDWEEL might be a potential HLA-A0201 restricted CTL epitope from hepatitis B virus X protein;further study is needed for verification of its immunity in vivo.

Objective To investigate the cytotoxic effects of IL-2 combined with different dosages of sorafenib on renal cellular carcinoma cell line 786-0. Methods Renal carcinoma cell 786-0 was cultured. Then , IL-2 (20 μmol/L) combined with different dosages of sorafenib (6.9, 13.8, 20.8 μmol/L) were used to treat tumor cell 786-0. The inhibitory effect on cell proliferation was determined by MTT assay. Cell apoptosis was measured by Annexin V-FITC kit. The tumor-bearing mice models were established and divided into four groups. Results The tumor cell growth was inhibited with the time-course correlation in all groups. In the 48-hour high doses group, the inhibitory rate was up to (74.67±1.87) %. The rates of cell proliferation inhibition and cell apoptosis were higher in the high dosages group than those in the other groups. Conclusions Immunotherapy combined with target therapy could significantly inhibit the growth of renal cellular carcinoma. But we should find a proper dosage, which could improve the clinical effect and reduce the adverse effect.