Objective To express functional haemegglutinin(HA)protein in two different bacularvirus expression systems.Methods The whole open reading frame of A/Sichuan/1/2009(H1N1)HA was obtained by synthesis,and the HA protein were expressed in insect cells by two different bacularvius expression systems:BaculoGold system and Bac-to-Bac system. Soluble HA protein was identified by Western blot and haemegglutination test. Results The correct full length of HA gene was obtained and cloned into pAcGP67B and pFAST Bacl vectors,respectively.After 3 rounds of virus amplifyjng by re-infection of Sf9 cells,the HA protein was detected in supematant of BaculoGold system and in intracellular of Bac-to-Bac system which is much better than the former.Purified HA protein was positive not only identified by Western blot,but also detected by haemegglutinin test. Conclusion Functional HA protein was successfully expressed in two distinct bacularvirus expression systems,of which the Bac-to-Bac bacularvirus expression system is more suitable for expression of A/Sichuan/1/2009(H1N1)HA protein.

Objective To study the effect of interleukin 2(IL-2)of clinical dose range,on the proliferation of human peripheral blood T cells,with special emphasis on the number and functional phenotype of γδT cells.Methods Human peripheral blood mononuelear cells(PBMCs)were isolated by density gradient centrifugation and cultured for 2 weeks at different IL-2 concentrations.Ratio and phenotype of different T cell subpopulations before and after in vitro expansion were explored by immunofluorescence staining.Cell number was estimated by trypan blue staining and cell counting.Results Within the four functional phenotypes of Vδ1 as well as Vδ2 γδT cells,CD27~+cells(including CD27~+CD45RA~+and CD27~+CD45RA~-subsets)expressed lymphoid tissue homing receptor CCR7,whereas CD27~-cells(including CD27~-CD45RA~+and CD27~-CD45RA~-subsets)had the peripheral tissue homing potential.All the studied γδT functional subsets showed the expression of activity related receptors,and the ability of a rapid production of various amount of cytotoxicity related effectors following mitogen stimulation.Although IL-2 at high concentration suppressed the proliferation of CD4 T cells,it may promote the proliferation of γδT cells.The proliferated γδT cells were mainly CD27~-CD45RA~-effector cells.Conclusion IL-2 of the clinical dose range may promote proliferation of human peripheral blood γδT cells,which might have important biological significance in IL-2 based anti-tumor therapy.

Objective To establish an expression system of TCRγ9/δ2-Fc protein by baculovirus vector ex-pression system and identify biological function of expressed TCRγ9/δ2-Fc protein. Methods γ9Fc and 82 (OT3) Fc gene fragments were amplified by overlap PCR and inserted into expression vector pBACp10ph. The recombinant plasmid pBACp10ph-γ9/δ2(OT3)-Fc and the baculovirus DNA were co-transfected into st9 cells. The expression position of TCRγ9/δ2 (OT3)-Fc was identified by Western blot and the expression efficiency of γ9Fc and δ2 (OT3) Fc was tested by flow cytometry (FCM). Furthermore, the binding activity of TCRγ9/δ2 (OT3)-Fc protein with SKOV3 ceils and MNS protein was evaluated with laser scanning confocal microslopy and surface plasmon resonance (SPR). Results The recombinant vector pBACp10ph-γ9/δ2(OT3)-Fc was constructed and TCRγ9/δ2(OT3)-Fc protein was expressed in sf9 ceils. However, the expression efficiency of γ9Fc and 82 (0T3) Fc was quite differ-ent. It was proved that purified TCRγ9/δ2 (OT3)-Fc protein can bind with SKOV3 cell and MNS protein. Conclu-sion TCRγ9/δ2-Fc protein is successfully expressed in baculovirus vector expression system and TCRγ9/δ2-Fc protein can simulate the binding activity of TCR in vitro.

Objective To identify high affinity ligand MULT1-transgenic mice,analyze its elementary phenotype,and to perform primary functional study in MULT1.Methods We identified the genotype of transgenic mice by PCR and detected transcription level of MULT1 in tails by real-time PCR.The flowcytometry and immunohisto-chemistry were used to analyze the expression of MULT1 in various tissues.Results 25 3'S-strain and 13 4'S-strain MULT1-transgenic mice were obtained and which contain high level of MULT1 transcripts.MULT1 protein is mainly expressed in intestinal intraepithelial lymphocytes (iIELs) and thymocytes but not on splenocytes.Moreover,the levels of MULT1 expression in iIELs and thymocytes are associated with age.Within iIELs,the percentage of CD8~+ Tcells decreased,while CD4~+ CD25~+ Treg cells increased,when compared with wild-type mouse.Conclu-sion We successfully produced MULT1-transgenic mice.MULT1 is possibly associated with thymic development and aging,as well as immunoregulation in mice.

Objective To explore the role of hMSH2, a novel endogenous tumor-associated pro-tein ligand recognized by Vγ9δ2 T cells, in innate anti-cervix cancer immunity .Methods hMSH2 that ex-pressed on the surface of cervical cancer cell line HeLa cells was blocked by specific antibody .Then the differences in their effects on Vγ9δ2 T cells before and after antibody blockage were evaluated by cytotoxicity of Vγ9δ2 T cells and cytokines secretion .The serum levels of hMSH2 in patients with cervical cancer were detected by ELISA .Distribution of hMSH2 in cervical cancer tissues was analyzed by TMA immunohisto-chemistry .Results Decreased cytotoxicity and IFN-γsecretion were observed in Vγ9δ2 T cells against He-La cells blocked with specific anti-hMSH2 antibody .Serum concentration of hMSH 2 in patients with cervical cancer was slightly higher than that of healthy control .Altered distribution pattern of hMSH 2 was observed in cervical cancer tissues .Conclusion hMSH2 is involved in Vγ9δ2 T cells-mediated innate anti-cervical cancer immunity by enhancing their cytotoxicity and IFN-γsecretion.

Objective:To construct of transfectant cells expressing ??TCR with specific CDR3 sequence.Methods:Specific CDR3 region sequence of DBS4.3,a known ??T cell clone,was inserted into ?9 and ?2 chain to substitute original CDR3 sequence using overlapping PCR method.After the full-length ?9 and ?2 chains were ligated with expression vector pREP7and pREP9 respectively,they were co-trasfected into a cell line of human Jurkat cells with TCR ? chain gene-defective mutant J.RT3-T3.5.When the transfectant cells expressing specific ??TCR were stimulated by antigen,production of IL-2 was detected by ELISA and Realtime PCR.Results:By ELISA and Realtime PCR,it was exhibited that the transfectant cells expressing DBS4.3 specific ??TCR secreted IL-2 under stimulation of iso-butylamine and anti ??TCR antibody.Conclusion:The transfectant Jurkat cells expressing ??TCR with specific CDR3 sequence are successfully constructed.It provides a platform for the research of recognition mechanism of ??TCR.

Objective Establishment and characterization of healthy donor's ??T cell clones.Methods ??T cells were cloned by limiting dilution after positive sorting with 60Co irradiated allogeneic PBMC as feeder cells.Flow cytometry analysis and molecular biology technique were then used to identify ??T cell clones.MTT assay was used to verify their proliferation after incubated with epitope peptides recognized by ??T cells.Results A ??T cell clone had been established.The subtype of this clone was V ?9 V ?2 without expression of CD4 and CD8.Further studies indicated that epitope peptide EP6 could not only specifically bind to ??T cell clone but also trigger the proliferation of ??T cell clone.Conclusion A healthy donor's ??T cell clone was successfully established,which laid a solid foundation for further study on ??T cells.

Objective To establish an expression system of TCR?9/?2-Fc protein by baculovirus vector expression system and identify biological function of expressed TCR?9/?2-Fc protein.Methods ?9Fc and ?2(OT3)Fc gene fragments were amplified by overlap PCR and inserted into expression vector pBACp10ph.The recombinant plasmid pBACp10ph-?9/?2(OT3)-Fc and the baculovirus DNA were co-transfected into sf9 cells.The expression position of TCR?9/?2(OT3)-Fc was identified by Western blot and the expression efficiency of ?9Fc and ?2(OT3)Fc was tested by flow cytometry(FCM).Furthermore,the binding activity of TCR?9/?2(OT3)-Fc protein with SKOV3 cells and MNS protein was evaluated with laser scanning confocal microslopy and surface plasmon resonance(SPR).Results The recombinant vector pBACp10ph-?9/?2(OT3)-Fc was constructed and TCR?9/?2(OT3)-Fc protein was expressed in sf9 cells.However,the expression efficiency of ?9Fc and ?2(OT3)Fc was quite different.It was proved that purified TCR?9/?2(OT3)-Fc protein can bind with SKOV3 cell and MNS protein.Conclusion TCR?9/?2-Fc protein is successfully expressed in baculovirus vector expression system and TCR?9/?2-Fc protein can simulate the binding activity of TCR in vitro.

Objective To study the relationship between the expression of NKG2D ligands and oxidative stress,and to analyze the effect of oxidative stress on the function of NK cells. Methods Tumor cells were cultured and exposed to hydrogen peroxide to develope an oxidative stress model. Then to detect the expression of NKG2D ligands in cells by Real-time PCR and Flow Cytometry. The cytotoxicity of NK cells to tumor cells was detected and compared by CCK-8 kit before and after oxidative stress. Results The expression of NKG2D ligands was induced by oxidative stress,however the NKG2D ligands induced was variable. The up-regulation of NKG2D ligands increased the cytotoxicity of NK cells efficiently,and this effect was blocked by anti-NKG2D antibody. Conclusion The expression of NKG2D ligands can be selectively induced by oxidative stress on tumor cells,and the improvement of the cytotoxicity of NK cells may enhance the immune responses accordingly.

Objective To study the role of human MutS homolog 2 (hMSH2), a newly identified protein ligand that was recognized by Vγ9δ2 T cells , in innate anti-gastric carcinoma immunity .Methods Flow cytometry and laser confocal microscopy were used to identify hMSH 2 that ectopically expressed on gas-tric carcinoma cell line 803.An anti-hMSH2 antibody was used to block hMSH2 to evaluate its effects on the cytotoxicity of Vγ9δ2 T cells and their cytokines secretion .Subcellular distribution of hMSH 2 in gastric car-cinoma tissues was examined by tissue microarray immunohistochemistry analysis .Results Ectopic mem-brane expression of hMSH 2 was observed on 803 cells at a relatively high level .Vγ9δ2 T cells blocked with specific anti-hMSH2 antibody showed a decreased cytotoxicity and a reduced IFN-γbut an increased TNF-αsecretion.Ectopic expression of hMSH2 was found in various types of gastric carcinoma tissues at different stages.Enhanced expression of hMSH2 was detected in specimens collected from patients with chronic super-ficial gastritis.Conclusion Ectopically expressed hMSH2 served as a stress-induced endogenous ligand which could promote the cytotoxicity of Vγ9δ2 T cells against gastric carcinoma cells and enhance their IFN-γsecretion.hMSH2 played an essential role in innate anti-gastric carcinoma immunity .