用分子杂交法检测实验小鼠体内柯萨基B组病毒RNA

DETECTION OF COXSACKIE B VIRUS RNA IN EXPERIMENTALLY INFECTED MICE BY MOLECULARHYBRIDIZATION

用来自柯萨基B组3型病毒基因组5′末端的cDNA克隆pCB111/51为探针,检测病毒感染小鼠动物模型中肠道病毒RNA。用柯萨基B组病毒CBU1和CBU2型分别攻击易感小白鼠乳鼠和成年鼠,可导致实验性急、慢性肌炎和急性心肌炎。冰冻和福尔马林固定、石蜡包埋组织标本,分别用于^(?)P标记探针槽印染膜杂交和生物素标记探针原位杂交。结果两种分子杂交方法都能从动物心肌炎、肌炎标本中检出柯萨基B组病毒RNA,并发现慢性肌炎持续存在长达6个月以上。 槽印染膜杂交技术具有结果客观、信号便于扫描定量等特点。生物素标记探针原位杂交结果与槽印染膜杂交法相一致,避免了同位素的缺点。并可以对病毒基因组进行细胞水平定位,有助于发病机理的研究。

Enteroviruses can cause verious human diseases. It has been noted that Coxsackie B viruses are etiologically related to myocarditis, myocitis and other diseases. In current study, a recombinant plasmid pCBⅢ/51, derived from 5, end sequence of Coxsackie B virus type 3 genome,were chosen as a probe to detect Coxsackie B virus RNA from experimentally infected mice. 32p-labeled probe was used in slot-blot hybridization to detect virus RNA quantitatively from the tissue samples, whils biotin-la-beled probe was employed in in situ hybridization to localise virus genome at the cellular level. Experimental myocarditis and myositis had been induced in mice and sukling mice by inoculation with Coxsackie B3 and B1 virus respectively. Frozen samples were prepared for the membrane hybridization and formalin fixed-parrafin embedded ones fori n situ hybridization. By either technique Coxsackie B virus RNA could be steadi- ty detected from myocarditis and myositis samples of exp rimental mice. It has been observed that chronic myositis persisted over six months.In slot blot hybridization, the hybridization index of two myocarditis and six myositis samples were higher than normal control ( >3×SD ). The result obtained by this technique is objective and quantitative. Formalin fixed,paraffin embedded thia sections were hybridized with biotin-labelled probe after pretreatment with proteinase K. The hybridization signals were visualized by HRP-OPD reaction and localized in muscular fibers of myocarditis and myositis. The signals were not observed within infiltrative lymphocytes. The result obtained by in situ hybridization is in agreement with slot blot hybridization. Morever,it avoids the weak points of radioactive probe and can localize virus genome at cellular level, which will faciliate research on pathogenesis