Objective To develop and verify a fluorescent quantitative polymerase chain reaction(qPCR)method based on TaqMan probe for the detection of E301R gene of African swine fever virus(ASFV),so as to apply the method to the detection of ASFV in clinical samples. Methods By analyzing the E301R gene sequence of ASFV,specific primers and TaqMan probes were designed for the conserved region of the gene. The fragment of E301R gene amplified by PCR was cloned into pCAGGS vector,and the standard plasmid was constructed. The qPCR detection method based on TaqMan probe was developed and verified for the linear range,precision,specificity,sensitivity and accuracy. The developed method was used to detect 92 clinical samples,and the results were compared with those detected by commercial real-time PCR diagnostic kit. In addition,the transcriptional dynamics of E301R gene was analyzed by using the developed method. Results In the range of 1. 6 ×（10~1-108）copies/μL,the standard plasmid showed a good linear relationship with Ct values,and the linear regression equation was:y =-3. 239 x + 40. 774,with the correlation coefficient of 0. 994. The CVs of repeatability and intermediate precision verification were both less than 2%. Except for ASFV,there was no amplification curve of classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),and porcine circovirus type 2(PCV2). The minimum detection limit was 1. 6 × 10~1copies/μL of standard plasmid. The spike recoveries of 1. 6 × 10~2and 1. 6 × 10~1copies/μL standard plasmid were between 90% and 110%. The coincidence rate of the detection results between the developed method and commercial real-time PCR diagnostic kit for 92 clinical samples was 96. 7%(Kappa = 0. 932,P = 1. 000). E301R gene may be the transcription gene in the middle stage of ASFV infection. Conclusion The developed detection method of qPCR based on TaqMan probe has good precision,specificity,sensitivity and accuracy which can be used for the detection of ASFV in clinical samples

We isolated polyA~+ mRNA direcdy from tumor tissue of ovarian carcinoma using Oligotex~(TM) Direct mRNA Kit (QIAGEN) to synthesize the first and second strand cDNA. The ds-cDNA termini were blunted with pfu DNA poly-rnerase. The blunted cDNAs were added EcoR I adaptor, and then were digested by Xho I . Small cDNA molecules(

Objective To explore the value of glypican-3(GPC-3)mRNA and paternally expressed 10(PEG10)mRNA in peripheral blood in diagnosis of metastasis in hepatocellular carcinoma(HCC). Methods With SYBR Green I as fluorescence signal,real-time fluorescent quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of GPC-3 mRNA and PEG10 mRNA in peripheral blood from patients with HCC with metastasis(n=8),HCC without metastasis(n=12)and hepatic cirrhosis(n=11),and receiver operator characteristics curve(ROC)and specific parameters were adopted to analyse their value in predictive and exclusive diagnosis. Results The expression of GPC-3 mRNA and PEG10 mRNA in HCC with metastasis was significantly higher than that in HCC without metastasis and in hepatic cirrhosis(P<0.05),while there was no significant difference in the expression of GPC-3 mRNA and PEG10 mRNA between HCC without metastasis and hepatic cirrhosis.In single test,the sensitivities in the differential diagnosis between HCC with metastasis and HCC without metastasis were 66.7%for GPC-3 mRNA and 72.2%for PEG10 mRNA,and the specificities were 91.7%and 91.7%.respectively.The areas under ROC were 0.748 for GPC-3 mRNA and 0.812 for PEG10 mRNA.With two markers in parallel test,the sensitivity,specificity,negative likelihood and diagnostic accuracy were 90.7%,84.O%,0.11 and 83.3%,respectively.In serial test,the sensitivity,specificity,positive likelihood and diagnostic accuracy were 60.5%,98.7%,45.5 and 73.3%,respectively. Conclusion Detection of GPC-3 mRNA and PEG10 mRNA in peripheral blood may help to predict blood metastasis and extrahepatic metastasis of HCC,and PEG10 mRNA works better than GPC-3 mRNA.The serial test of GPC-3 mRNA and PEG10 mRNA is helpful to the predictive diagnosis of peripheral blood metastasis of HCC.

Objective:To explore whether apoptotic ovarian cancer cells induced by chemotherapy drugs paclitaxel and cisplatin can be cross-presented by dendritic cells(DCs) and enhance immune responses.Methods:DCs were induced from peripheral blood monocytes cells by GM-CSF/IL-4 for 6 d,then they were stimulated with either apoptotic ovarian cancer HO8910 cells,frozen-thawed HO8910 cells or control cells for 4 h.Their surface markers and phagocytotic ability were detected by flow cytometry and confocal microscopic scanning assay,respectively.DCs of different groups were cultured with CD8+ T cells isolated by magnetic cell sorting,and the ability of DCs to activate CD8+ T cells was evaluated by 3H-TdR,the activity of CTL to kill tumor cells was evaluated by LDH.Production of IFN-? by CD8+ T cells was measured by ELISPOT.Results:Apoptotic ovarian cancer cells induced by chemotherapy drugs paclitaxel and cisplatin could be phagocytized by DCs,which subsequently promoted the maturation and antigen presenting ability of DCs.Apoptotic ovarian cancer cells implused DCs significantly promoted proliferation of CD8+ T cells compared with that of control cells(P

Objective: Full expression of class I HLA molecules on tumor cells is pivotal in priming the tumor antigen-specific CTLs for effective iramunotherapies. Tumor cells display abnonnal expression of HLA class I molecules in human ovarian carcinoma. Methods: In this study, the expression of class I molecules in human ovarian cancer cells was determined by using Western bloting, immunohistochemistry testing and flow cytometry. The mRNA of TAP (transporter associated with antigen processing) and LMP(low molecular weight polypeptide) were exannined at the same time by RT-PCR. Results: It has been shown that class I molecules were unable to be, or only weakly, expressed in five of eight ovarian cancer cell lines. On these cell lines abnormal in class I expression, no or only little mRNA were detected for TAP1, TAP2, LMP2 and(or) LMP7 genes. Class I expression, however, could be partially recovered by incubating the tumor cells at 25℃ when compared with those at 37℃ . Conclusion: The results suggested that the dysfunction of TAP and IMP genes, following by a defective expression of class I molecules, might be one of the mechanisms enable ovarian cancer cells to escape from immune surveillance.

Objective:To explore the characteristics of human melanoma-specific antigen peptides by HLA-A2 restricted tumor-infiltrating lymphocytes.Methods:The HLA-A2 protein and polypeptides molecules were purified from the three tumor cell lines(624-Mel, Chap-Mel and JY) by immunoaffinity chromatography, after the peptides bound to HLA-A2 protein solution were acidified with acetic acid and boiled by high temperature, and centrifuged through an Ultra-CL filter, then the peptides extracts were fractionated by revered phase high pressure liquid chromatography(RP-HPLC). Individual fractions were assessed for their ability to reconstitute melanoma-specific epitopes by adding to the HLA-A2 Ag-procceing mutant cell, T2. The biological feature of one of three active peptides from RT-HPLC samples was performed by mass spectrometric analysis. The synthetic peptides identical to active peptide sequences were determined in the reconstitute test.Results:Three prominent peaks(P19, P25 and P31) of the fraction from 624-Mel were observed in the reconstitute test, TIL killing rate was 67% for (P31) peptide fraction. The mass spectrometric analysis of one of active peptides (P31) showed that at mass-to-charge ratio(m/z) 948 has been usually nine residues. The sequence is H+ Ala Lue Trp Lue Phe Phe Gly Val Lue OH-. The peptide synthesized comprising epitopes were verified.Conclusion:These results showed the peptides derived from active fractions were related to human melanoma-specific tumor antigen peptides recognized by HLA-A2-restriced TIL. These peptides could develop novel peptide-based an anti-tumor vaccine for immunotherapy of CTL.

Objective:As the function of ??T cells and NK in the immunological therapy is showing more and more important,the characteristics of ?? T cells,NK and LAK were analyzed comparatively after that they were richened by isolating and incubating in vitro.Methods:The cells were collected using MACS after the cells were panned respectively with special monoclonal antibodies.Then the characteristics including proliferation,phenotype,cytotoxin and the down regulation blocked by specific antibodies were analyzed.Results:The ?? T cells can expand 600～800 times after culturing 2 weeks and the percent of CD3,CD8 and ?? expressed on the collected cells were 72.29%,58.02% and 65.98% respectively ?? T cells showed the high cytotoxin to K562(NK sensitive cell line),Raji and XG 7 cell lines(NK non sensitive cell lines).The percentage of cytotoxin reached 35.98%,52.27% and 69.08% respectively compared with ??T respectively.No obviously change of percent of ??T cytotxic ability to these target cells were observed using special MHC class I monoclonal antibody to block the ??T cell before coculturing the target cells with ?? T cells.Conclusion:All of ?? T cells,NK and LAK showed the non specific cytotoxin to tumor cells.The cytotoxic capability of ?? T cells did not be effected after blocking with MHC class I monoclonal antibody.These results demonstrated that ?? T cell could kill more kinds of tumor cells than NK and LAK.

Objective:To clone HLA A *0201 cDNA and express it transiently on COS 7 cells.Methods:HLA A cDNA isolated from the HLA A *0201 positive lymphocytes by using RT PCR was cloned into pBluescript II SK vector directionally.After the sequence was confirmed,the cDNA was inserted into mammalian expression vector pcDNA3.The recombinant vector was transfected into COS 7 cells by cation liposome.The transient expression on the cells was measured by flow cyteometer.Results:The cDNA was identical with HLA A *0201 cDNA published on Genebank.Determined by flow cytemeter,the expressing rate was recorded for 57%.Conclusion:We have cloned HLA A *0201 successfully and expressed it transiently on COS 7 cell,which would be potentially useful in research on killing tumor restricted by HLA A2.

Anemia, Sideroblastic ; genetics ; Calreticulin ; genetics ; Humans ; Mutation ; Myelodysplastic-Myeloproliferative Diseases ; genetics ; Phosphoproteins ; genetics ; RNA Splicing Factors ; Ribonucleoprotein, U2 Small Nuclear ; genetics

AIM:To explore the inhibitory effects of tumor associated mitochondrial protein 12 (TAMP12) on tumor cell apoptosis. METHODS: (1) A retrovirus expression vector was recombinated and transfected into the packaging cell line PA317. The virus particles were obtained to infect the target cell line HepG2 low expressing of TAMP12. The expression of TAMP12 mRNA was detected by RT-PCR. The subcellular localization and quantification of TAMP12 protein labeled with double fluorescein were observed under confocal laser scanning microscope (CLSM). (2) Hoechst33258 staining and flow cytometry (FACS) were used to analysis the apoptosis of HepG2 cells treated with 5-fluorouracil (5-FU). RESULTS: (1) The CLSM observation showed that TAMP12 protein was mainly expressed in mitochondria of HepG2 cells. The expressions of TAMP12 gene and protein were stable and high in transfected HepG2 cells. (2) Upon treatment with 5-FU, the transfected HepG2 cells showed a fairly integrated nucelus while the control HepG2 cells exhibited chromatin condensation, marginalization and karyorhexix. Moreover, the apoptosis rate of transfeced HepG2 cells was significantly lower than that in control HepG2 cells (P