HBV耐药相关DNA突变体的构建及在寡核苷酸基因芯片分析中的应用

拉米呋啶（Lamivudine)是近年来开发成功的一种治疗HBV慢性感染病人的核苷类药物。随着拉米呋啶在临床上广泛使用，HBV耐药现象已成为临床实践中的棘手问题。建立HBV耐药测定技术成为HBV基础研究和临床实践中企待解决的一个重要问题。寡核苷酸芯片（Oligochip)是近年来发展并逐步成熟的一种高通量基因检测技术，已成功应用于基因突变快速和高通量检测。本研究在构建HBV拉米呋啶抗性相关HBV多聚酶基因突变体基础上，设计并制备了HBV耐药寡核苷酸芯片（HBV－Lam Oligochip)。根据HBV拉米呋啶抗药性相关突变主要位于HBV多聚酶基因的L526、A546、M550和V553等氨基酸位点，设计了28条寡核苷酸探针。探针对应序列为HBV DNA聚合酶基因反义链，HBVHBVhb长度为15－18nt。探针合成时在其3′端接氨基和间隔臂（spacer)等特定修饰；探针纯化与定量后，用基因芯片点样仪点到醛基修饰载玻片上，制成HBV耐药寡核苷酸芯片（HBV－Lam Oligochip)。为了分析基因芯片的性能，克隆了833号HBV病人血清中HBV DNA聚合酶基因，测序证实为HBV DNA野生型（即未发生突变）。以该DNA为模板，用PCR法构建了HBV耐药相关突变体。用HBV－Lam Oligochip对HBV野生型DNA和人工构建DNA突变体进行了检测。结果发现，HBV－Lam Oligochip能有效检测出野生型DNA序列。检测突变体时，HBV－Lam Oligochip检测结果与相应突变体DNA序列一致,表明HBV－Lam Oligochip不仅可检测出野生型序列，而且可有效地分辨出单碱基突变，可应用于HBV耐药基因突变临床检测。

Preparation of HBV Lamivudine related DNA mutants and application for property analysis of the oligochips for genetic detection of HBV Lamivudine related resistance

Lamivudine(3TC),an anti-HIV nucleotide analog,is a promising drug developed in recent year for treat-ing of HBV persistent patient.With clinically wide application,HBV resistance to lamivudine become a problemic is sue,which re strict effective application of the drug.Developing novel methods for HBV lamivudine resistance detec-tion plays an im portant role in HBV basic re search and clinical practice.Oligochip is one of the genetic tools devel-oped in recent years showing promising for gene mutation analysis in rapid and high-throughtput manner.For prapa-ration of an oligochip for detection of HBV lamivudine associated resis tance,28oligo probes(15-18nt,but most were17nt)were designed and sy hthesised according to the published hot point muta tions in related to HBV lamivudine resis tance,and then spotted by mi croarrayer to aldehyde modified glass slide to fabricate the HBV-Lam Oligochip.For analysis the property of the HBV-lam Oligochip,4mutants of HBV DNA polynerase containing the hot muta-tions which were associated HBV lamivudine resistance were con structed in vitro by PCR based on the wild type HBV DNA polymerase sequence from No.833HBV positive serum.The HBV-Lam Ologochip can effectively detectthewildtypesequenceofHBVDNApolymerase,butalsosufficientlydiscriminatethepointmutationinthesemu-tantsinrelatedtoHBVlamivudineresistance.WhenusedfordetectionofHBVclinicalsample,thesequencesin-ducedfromthehybridizationsignalsoftheHBV-LamOlogochipwerethesameofthatfromHBVDNAsequencing.TheseresultssuggestthatHBV-LamOligochipisaneffectivegenetictoolusingfordetectionofHBVlamivudineresistance,andhasgreatpotentialforevaluatingtheeffectofHBVtreatment,anddirectingclinicallyrationaldrugusefulness.