Objective:To construct His-tagged peptide of human STAT4 (565-748 amino acid) expression vector and induce its expression in Escherichia coli,followed by purification.Methods:STAT4 gene fragment encoding C-terminal peptide of 565-748 amino acid was amplified by PCR using pEGFP-STAT4 as the template.The PCR product was inserted into prokaryotic expression vector pET-28a and was transformed into component E.coli BL21 cells.By isopropyl-β-D-thiogalactoside(IPTG) induction,fusion protein was found to be expressed in the inclusion body and was denatured by using the urea denaturation buffer followed by renaturation and purifi-cation.Finally the purified protein was confirmed by Western blot.Results:The STAT4 truncated gene encoding 565-748 amino acids peptide was amplified by PCR and inserted into pET-28a vector.After the recombined plasmid was transformed into component BL21, the His-tagged-STAT4 (565-748 amino acids) fusion protein was induced and obtained after denaturation,refolding,purification and dialysis.Conclusion:The eukaryotic expression vector containing the truncated human STAT4 gene encoding 565-748aa peptide has been successfully constructed and the fusion protein was obtained.

OBJECTIVE:Interferon-γ (IFN-γ) and signal transducers and activators of transcription (STATs) each play an important role in carcinogenesis associated with viral infection. Cervical cancer is almost invariably associated with infection by human papillomavirus (HPV), and previous studies suggested that dysregulation of the signal pathway involved in IFN-γ and STATs is associated. Our objective was to evaluate the association of SNPs in STAT2, STAT3, and IFN-γ with cervical cancer susceptibility in Chinese Han women in Hunan province.MATERIALS AND METHODS:Genomic DNA was extracted from peripheral blood samples of 234 cervical cancer patients and 216 healthy female controls. STAT2 and STAT3 genotyping was performed using polymerase chain reaction-restriction enzyme (PCR-RE) analysis. IFN-γ genotyping was detected by PCR-amplification of specific allele (PASA).RESULTS:For STAT2 rs2066807 polymorphisms, there was no significant difference of genotype distribution (P=0.827) and allele frequencies (P=0.830, OR=1.09, 95% CI: 0.51-2.31) between cases and controls. For STAT3 rs957970 polymorphisms, there was no significant difference of genotype distribution (P=0.455) and allele frequencies (P=0.560, OR=0.92, 95% CI: 0.71-1.20) between cases and controls. For IFN-γ +874A/T polymorphisms, there was no significant difference of genotype distribution (P=0.652) and allele frequencies (P=0.527, OR=1.12, 95% CI: 0.79-1.59) between cases and controls.CONCLUSION:These results suggest that polymorphisms in STAT2, STAT3 and IFN-γ genes are not likely to be strong predictors of cervical cancer in Han women in southern China.

Objective: In order to detect two Signal Nucleotide Polymorphisms(SNPs),rs12422499 and rs2066807 gene of Signal Transducer and Activator of Transcription Factor 2(STAT2),a simple and reliable method,named polymerase chain reaction-primer introduced restriction analysis(PCR-PIRA) was established.Methods: DNA from peripheral blood was extracted using the trace salting-out method.Design PCR primers to amplify the gene fragment containing STAT2 SNP,rs2066807 or rs12422499,respectively.Digest the corresponding PCR product by PsyI or BseDII.Separate the digested products by electrophoresis.And then,the PCR products were sequenced.Results: The length of PCR product of the gene fragment containing rs2066807 was 469 bp.This product was digested by PsyI following by electrophoresis.According to the electrophretic maps,three genotypes of rs2066807 which were C/C(469 bp),G/G(262 bp and 207 bp) and C/G(469 bp,262 bp and 207 bp) were decided.The length of PCR product of the gene fragment containing rs12422499 was 189 bp.This product was digested by BseDI following by electrophoresis.According to the electrophretic maps,three genotypes of rs12422499 which were C/C(189 bp),G/G(134 bp and 55 bp),and C/G(189 bp,134 bp and 55 bp) were decided,The PCR products were sequenced.The sequencing results of the two SNPs were in good agreement with that detected by PCR-PIRA.Conclusion: The PCR-PIRA method for detection of SNPs of STAT2 was established.The procedure of PCR-PIRA is very simple and convenient,only including a PCR amplification followed by digestion by an enzyme.The sequencing results of the two SNPs were consistent with that detected by PCR-PIRA,demonstrating that PCR-RIPA is reliable.The strategy of PCR-PIRA does not only offer an experimental method for detection of polymorphism of STAT2,but also a mean of gene point mutation.

【Objective】To construct the(signal transducer and activator of transcription 4,STAT4) eukaryotic expression plasmid and express it in HEK293 cells.【Methods】STAT4 cDNA was amplified from human peripherial blood mononuclear cells(PBMC) by reverse-transcription polymerase chain reaction(RT-PCR) and inserted into the pEGFP-C1 vector digested by SalI/BamHI for construction of pEGFP-STAT4 plasmid.This plasmid was identified by sequencing as well as double enzymes digestion.pEGFP-STAT4 was transfected into HEK293 cells.The fusion protein EGFP-STAT4 was detected by fluorescence microscope and Western Blot.【Results】2 247 bp fragment of STAT4 cDNA full-length sequence was cloned by RT-PCR,and it was consistent with the known STAT4 sequence in GenBank.After the plasmid pEGFP-STAT4 was transfected into HEK293 cells,the fusion protein GFPSTAT4 was instantaneously expressed in some of cells.【Conclusion】The recombined pEGFP-STAT4 eukaryotic expression plasmid is successfully constructed and instantaneously expressed in HEK293 cells.