Fusion Protein Expression And Affinity Purification Of MTB His-LprG-FLAG In HEK293T Cells And Genetic Polymorphisms In Interleukin-23R Gene And Its Association With Pulmonary Tuberculosis In Eastern China

Objective To construct an eukaryotic expressing vector of the Mycobacterium tuberculosis lipoprotein G (LprG) and to express and purify the recombinant protein by affinity chromatography.Methods The LprG gene was amplified by PCR from the genome of strain H37Rv. The subsequent PCR product and eukaryotic expressing vector pcDNA3.1 were digested by certain restriction enzymes. The recombinant vector, pcDNA3.1-His-LprG-FLAG, was constructed by ligation of target genes and the vector. After identified by enzymes digestion and DNA sequencing analysis, the correct recombinant vector was applied for transfection of HEK293T cells. The target fusion protein successfully expressed in HEK293T cells was purified by Ni2+ affinity chromatography, and the purification and concentration of such protein was evaluated by SDS-PAGE and Western blotting.Results Such recombinatant vector, pcDNA3.1-His-LprG-FLAG, was successfully constructed, which was identified by double enzyme digestion and DNA sequencing. The fusion protein expressed in HEK293T cells with transfection of the vector could be indicated by Western blotting at molecular mass of 27 000. Under this framework of expression, the fusion protein could be prepared with ideal purification and concentration, evlatued by SDS-PAGE and Western blotting.Conclusion His-LprG-FLAG fusion protein was successfully expressed in HEK293T cells and purified.Objectives The aim of this study is to determine whether interleukin-23 receptor (IL-23R) polymorphisms are associated with PTB through a case-control study.Methods One polymorphism in the IL-23R gene (rs 11805303) were examined in 211 PTB patients and 200 healthy controls using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.DNA sequencing was used to validate genotype results.The application online software tested Hardy-Weinberg (HWE).Use the chi-square analysis genotype and allele frequency distribution in the case group and control group differences.Logistic regression was applied to estimate odds ratio (OR) and 95%confidence interval (95%CI) by adjustment for confounding factors, gender and age, thereby screening susceptibility or independent protection of genotype and allele.Use variance analysis to test differences of serum ghrelin between control group and case group.The results were analyzed by SPSS 16.0 software and haplotye analysis was conducted by SHEsis software.Results1. Genotype of IL-23 gene rs11805303 site in case group and control group were consistent with Hardy-Weinberg equilibrium,the samples were representative and comparable(P>0.05).2. There was no significant difference in the genotype and allele frequencies of IL-23R rsl 1805303 polymorphism between PTB patients and healthy controls. However, the data revealed that subjects with the rs11805303 CT genotype have a higher susceptibility to PTB [odds ratio (OR) = 1.966, 95 %confidence interval (CI) =1.177-3.286, P = 0.01]. IL-23R rs11805303 polymorphisms appeared to be irrelevant to PTB.3. After stratified by the gender,in the male group, subjects with the rs11805303 CT genotype have a higher susceptibility to PTB. IL-23R rs11805303 CC?TT?C and T was no associated with susceptibility to PTB.Also, in the female group, IL-23R rs11805303 CC?CT?TT?C and T was no associated with susceptibility to PTB.Conclusion1. In conclusion, the IL-23R rs11805303 CT genotypes might be a risk factor for PTB in eastern China. Further investigations with a larger sample size may be required to validate the genetic effects of IL-23R polymorphisms on PTB.2. After stratified by the gender,in the male group, IL-23R rsl 1805303 CC?CT? TT?C and T was no associated with susceptibility to PTB. Also, in the female group, IL-23R rsl 1805303 CC?CT?TT?C and T was no associated with susceptibility to PTB.