Cloning and expression of human filaggrin gene and detection of anti-filaggrin antibodies for diagnosis of rheumatoid arthritis / 中华检验医学杂志

Objective To construct the recombinant plasmid containing human filaggrin gene,purify and identify the immunoreactivity of the recombinant protein,and establish the indirect ELISA to detect AFA for diagnosis of RA.Methods The constructed plasmids were transformed into E. Coli Rosettagami(DE3).This fusion protein was purified by NAT chromatography.ELISA coated with the fusion protein Was established to detect the AFA in serum of patients,which included 114 cases of RA,56 cases of SLE,32 cases of OA and 40 cases of normal controls. The correlation between the results of AFA and anti-CCP in RA group were compared. Results 321 bp fragment of filaggrin gene was amplified and the recombinant expression vector pET-28a( + )-filaggrin was constructed. The sequence of filaggrin gene was the same as the sequence reported in the literatures. The Rosetta-gami (DE3) strains of E. Coli with recombinant vector showed high level of filaggrin protein after induction. The SDS-PAGE showed that the plasmid expressed the filaggrin fusion protein with molecule weight of 14 000 Da. The expression protein could be purified by Ni-NAT with activity. The absorbance value of AFA in RA group was 0.473 ±0. 248 while they were 0. 160 0. 088, 0. 121±0. 070, 0.050 0. ±018 in SLE, OA and normal groups respectively. There were significant differences of absorbance values of AFA between RA and SLE, OA, control group (t = 12.004, 14. 464, 18.078, P<0. 01, respectively). The positivities of anti-filaggrin in RA, SLE and OA were 48.2%, 5.4% and 3. 1% respectively. The positivities of AFA were significantly different between RA, OA and normal control groups (x~2 = 67. 088, P < 0. 01). There was positive correlation of results between AFA and anti-CCP antibody (r = 0.42, P < 0. 05 ) . The consistency rate of results between AFA and anti-CCP was 70. 1%. Anti-CCP was negative in 10 out of 114 patients with AFA positive. AFA can be used to diagnose RA with sensitivity of 48. 2% , specificity of 96.9% , positive predictive value of 93. 2% and negative predictive values of 67. 9% . Conclusions The purified human filaggrin fusion protein is successfully purified. The indirect ELISA method based on the recombinant protein shows good sensitivity and specificity. Joint detection with AFA and anti-CCP can improve the positive rate of detection.

Prediction of CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains in China / 中华微生物学和免疫学杂志

Objective To predict the CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains, which were prevailing in China. Methods Total of 236 plasma samples were from the 3rd National HIV Molecular Epidemic Survey (NMES3). All the subjects were infected with HIV-1 CRF07_BC viruses. The tat exon 1 region was amplified by reverse transcription reaction and nested polymerase chain reaction (nested-PCR), then the PCR products were sequenced. The distribution of CTL epitopes of this region were predicted by on-line software BIMAS HLA Peptide Binding Predictions and statistics software. Results To-tal of 236 CRF07_BC strains were from 16 provinces, mainly in intravenous drug asers(58.9%)and then sex(25.0%). It was showed that there were 12 CTL epitopes of 236 Tat exon 1 region of CRF07_BC strains mainly located in proline-rich region, cysteine-rich region and core-region. Those epitopes were banded by 5 HLA presenting molecules in genotype(A * 2501 ,A * 2902, B * 15,B * 5301 and Cw * 1203) and 6 HLA presenting molecules in serotype (B53, B58 ,B57 ,A3 ,A68 and Cw12). The frequency of single amino acid substitution was more than 50% in 7 CTL epitopes. Conclusion The CTL epitopes in Tat exon 1 of CRF07 _BC strains were located in different functional regions, and there were some amino acid variations in them.

The effects of transfection of CXCL10 gene into breast cancer cell line MCF-7 on expression of tumor related genes / 中华检验医学杂志

s were 0.573±0.016, 0.707±0.008 and 0.711±0.013). Conclusions CXCL10 can express stablely in MCF-7 cell lines, which resulted in down-regulation of expression of VEGF and STAT3 gene. CXCL10 played an important role in anti-tumor effect.

Soluble expression of a CXCL10-loop3-EGF fusion protein and its anti-tumor activity / 中国病理生理杂志

AIM: To evaluate the implication of CXCL10-loop3-EGF fusion protein for the activities of targeting tumor and anti-angiopoiesis. METHODS: RT-PCR was preformed to amplify CXCL10 coding sequence from PBMC activated by IFN-γ. CXCL10-loop3-EGF fusion gene, which was conducted by Over-Lap Extention PCR, was hinged up with plasmid pTG19-T, transfected to E. coli DH5α and processed positive colony selection. After ligated with plasmid pET32a(+), recombinant CXCL10-loop3-EGF fusion gene was then transfected to E. coli Origami B (DE3) and induced to express its coding fusion protein his-CXCL10-loop3-EGF. The recombinant fusion protein CXCL10-loop3 -EGF was purified by His-bind affinity chromatograph, enterokinase cleavage, ultrafiltration and dislysis. The transwell chemotatic test and HUVEC angiopoiesis inhibition test were performed to determine the anti-tumor responses and anti-angiopoiesis activity of CXCL10-loop3-EGF fusion protein. RESULTS: CXCL10-loop3-EGF fusion protein was successfully constructed and confirmed by SDS-PAGE analysis and Western blotting. Significant PBMC chematatic activity and HUVEC anti-angiopoiesis activity were observed. CONCLUSION: CXCL10-loop3-EGF fusion protein, which has perfect anti-tumor activity, is successfully constructed.

Immunologic mechanism of CXCL10 and its receptor involved in endometriosis / 中国病理生理杂志

AIM: To investigate the immunologic mechanism of CXC chemokine ligand 10(CXCL10) and its receptor CXC chemokine receptor 3 (CXCR3 ) involved in the process of endometriosis (EM). METHODS: Serum samples were collected from 3 groups; EM patients without operation (n = 76) , EM patients with operation (n = 10) and the normal control persons (n =76). CXCL10 and CA12S concentrations were detected by means of ELISA and chemilumino-metry. Cell surface antigens on the activated PBMC - CD3 and CXCR3, as well as CXCR3 subgene - CXCR3A and CX-CR3B were tested by flow cytometry (FC) and RT - PCR when PBMC was separated from women with EM ( n = 10) and without EM (n = 10), and then activated. RESULTS: Serum CXCL10 concentrations between three groups were signifi-canly different (P < 0.05). Compared to normal control group, although the supernatant CXCL10 concentration and CD3~+ /CXCR3~+ PBMC number in EM group has no significant difference (P >0.05) , highly expressed CXCR3B in EM group rather than CXCR3A was observed. CONCLUSION: CXCL10 in women with EM is low, indicating that it plays a vital role in the process of EM and immune system of the women with EM is defected and impaired. The immunoreactivity of PBMC from both EM patients and normal person is same to activated signal, but the productions are different: PBMC in EM group mainly express CXCR3B but PBMC in normal person mainly express CXCR3A after activation, which may be one of the immune mechanisms that EM escapes from immunological lethal effect of the infected host.

Identification of recombinant baculovirus and determination of virus titer with fluorescence quantitative PCR assay / 中国病理生理杂志

AIM: To develop a real - time PCR assay based on TaqMan technology for the identification of recombinant baculovirus and determination of virus physical titers in Bac - to - Bac system. METHODS: The recombinant baculovirus containing human IL- 18 gene was produced using Bac -to- Bac system. A 10 -fold serially diluted primary viral stock was used for plaque assay and DNA extraction. Bacmid (baculovirus plasmid) was 10 -fold serially diluted and served as standards. Real - time PCR amplification of the IL - 18 gene was performed in triplicate for each diluted recombinant virus. At the same time, plaque assays were performed using overlay agarose method. RESULTS: The standard linear range (101 to 108 copies) for quantitation was achieved with the standard curve. We also find that the"vg/mL"titer value is generally about 10 times than"pfu/mL"titer of the same recombinant virus stock. CONCLUSION: A TaqMan real -time PCR method is established to identify the recombinant baculovirus and determine the"vg/mL"titer of virus. The method is rapid and quantitative over a wide range of virus titers.

Identification of recombination baculovirus and determination of virus titer with fluorescence quantitative PCR assay / 中国免疫学杂志

Objective:To develop a real-time PCR assays based on TaqMan chemistry for the identification of recombinant baculovirus and determination of virus physical titers in Bac-to-Bac system.Methods:The recombinant baculovirus containing human IL-18 gene was produced using Bac-to-Bac system.A 10-fold series diluted primary viral stocks were used for plaque assay and DNA extraction.Bacmid(baculovirus plasmid) was 10-fold series diluted and served as standards.Real-time PCR amplification of the IL-18 gene was performed in triplicate for each diluted recombinant virus.At the same time,plaque assays were performed using overlay agarose method.Results:The standard linear(101 to 108 copies) from quantitation was achieved with the standard curve.We also find that the "vg/ml" titer value is generally about 10 times than "pfu/ml" titer of the same recombinant virus stock.Conclusion:A TaqMan real-time PCR method is established to identify the recombinant baculovirus and determine the "vg/ml" titer of virus.The method is rapid and quantitative over a wide range of virus titers.

Expression and characterization of an EGF-IL-18 fusion protein for targeting (therapy) in insect cells / 中国免疫学杂志

Objective:To express and purify a new fusion protein harboring human epidermal growth factor receptor(EGFR) binding domain and Interleukin-18(IL-18), as well as preliminary assay biological activity of recombinant proteins.Methods:Fusion protein was expressed in insect cells Spodoptera frugiperda cell (Sf9) by using Bac-to-Bac system, and an abbreviation purification procedure was used to purify fusion protein. IFN-?induction assay and EGF Receptor competitive test was used to determine fusion protein's biological activity.Results:SDS-PAGE and western blot assay showed that purified EGF-IL-18 fusion protein had high purity in 20 kD as expected and had the same antigenic specificity as human IL-18. IFN-?induction assay and EGF Receptor competitive test showed that fusion protein induced production of IFN-?in human PBMC and bound with tumor cells.Conclusion:EGF-IL-18 fusion protein has been successfully expressed and purified in insect cells and shows potential to apply in targeting therapy for tumor.