不同耐药型的结核分枝杆菌hspX基因序列与表达的比较

Comparison of hspX gene sequence and expression in Mycobacterium tuberculosis with different drug resistance types

目的比较不同耐药型的结核分枝杆菌(MTB)热休克蛋白基因hspX序列与表达,初步探讨hspX与MTB的耐药是否存在相关性。方法选取4种一线抗结核药物全部敏感株(S)、同时对异烟肼和利福平耐药(MDR)和一种耐药[单耐利福平(MTB^(R+))、单耐异烟肼(MTB^(H+))、单耐链霉素(MTB^(S+))和单耐乙胺丁醇(MTB^(E+))]的菌株各10株,采用改良的十二烷基苯磺酸钠裂解法和TRIzol法分别提取各组菌株的DNA和RNA,使用特异性引物进行PCR和实时荧光定量PCR确定hspX基因的存在及其表达。对PCR产物进行测序以检查正向和反向的多态性,并用DNAman软件进行与标准株H37Rv序列比对。以敏感菌株(S)组为对照组进行两两对比分析不同耐药型的MTB菌株hspX表达的差异。结果PCR成功获得各组特异性的hspX基因,与H37Rv序列比对具有97%~99%的同一性,无存在明显差异;以相对定量2^(-ΔΔCt)法计算获得S、MDR、MTB^(R+)、MTB^(H+)、MTB^(S+)和MTB^(E+)菌株hspX的表达分别为0.98(0.89,1.23)、1.49(1.10,2.04)、1.41(0.55,2.80)、1.37(0.68,2.71)、0.91(0.59,1.62)和0.70(0.48,1.18)。以敏感菌株(S)组为对照组,不同耐药型菌株组与其比较,hspX表达差异均无统计学意义(P均>0.05)。结论hspX基因在MTB中是保守的。在自然状态下,hspX基因在MTB的耐药性方面无明显相关性。

Objective To preliminarily explore whether there is a correlation between heat shock protein gene hspX and drug resistance of Mycobacterium tuberculosis(MTB)by comparing the sequence and expression of hspX in MTB with different drug resistance types.Methods Ten strains sensitive to all four first-line anti-tuberculosis drugs(S),one drug resistance(rifampicin-resistant MTB^(5+),isoniazid-resistant MTB^(E+),streptomycin-resistant MTBS+and ethambutol-resistant MTB^(E+))and multidrug resistance(MDR)to isoniazid and rifampicin were selected,respectively.The DNA and RNA of strains in each group were extracted by modified SDS lysis method and TRIzol method,respectively.The existence and expression of hspX gene were determined by PCR and RT-PCR using specific primers.PCR products were sequenced to check the forward and reverse polymorphism,and DNAman software was employed to compare the sequence with the standard strain H37Rv.The difference in the expression of hspX among MTB strains with different drug resistance types was analyzed by pairwise with the sensitive strain group as the control group.Results The specific hspX gene was successfully obtained by PCR.The hspX gene sequence was 97%-99%identical with H37Rv,and there was no significant difference.The mean expression level of hspX through relative quantitation 2^(-ΔΔCt)in S,MDR,MTB^(R+),MTB^(H+),MTB^(S+)and MTB^(E+)strains was calculated as 0.98(0.89,1.23),1.49(1.10,2.04),1.41(0.55,2.80),1.37(0.68,2.71),0.91(0.59,1.62)and 0.70(0.48,1.18),respectively.There was no significant diff erence in the expression of hspX among diff erent drug-resistant strains groups when the sensitive strain group was regarded as the control group(all P>0.05).Conclusion The hspX gene is conserved in MTB.In natural state,there is no significant correlation between hspX gene and drug resistance of MTB.