摘要
目的:构建携带IHH WT和E95K、E95G、△E95、D100E突变体的RCAS病毒载体,并验证其有效性;优化RCAS病毒收集方法。方法:PCR扩增得到人IHH基因,采用原位杂交和半定量PCR的方法来验证上述载体能否过表达IHH mRNA;采用细胞裂解法收集RCAS-GFP病毒,通过鸡胚注射、冰冻切片观察的方法了解鸡胚感染情况。结果:构建的上述RCAS病毒载体在鸡胚细胞成功表达;优化了RCAS病毒收集方法,使最终滴度达108IU/ml。结论:该研究为在鸡胚肢芽中过表达不同IHH突变体蛋白打好了基础。
Objective： To construct RCAS vectors carrying IHH WT and E95 K, E95G, ZX E95, D100E mutants and prove its effectiveness; To optimize the method of virus collection. Method： IHH gene was amplified by PCR. To verify whether the constructed RCAS vectors can overexpres.s corresponding IHH mutant mRNA, in situ hybridization and semi - quantitative RT - PCR were used. Virus was collected through cell lysate, which was tested by chick embryos injection. Result：The constructed RCAS vectors were proved successful to overex- press IHH mRNA and the titer of RCAS was reached 108IU/ml. Conclusion：The work laid a solid foundation for overexpression of IHH mutant protein in the limb bud of chick embryo.
出处
《生物技术》
CAS
CSCD
北大核心
2013年第2期13-18,共6页
Biotechnology
基金
高等学校博士学科点专项科研基金项目(No.20090073120001)资助
