鸡包涵体肝炎病毒DNA探针的制备及其原位杂交的应用

The preparation of the DNA probe of chicken inclusion body hepatitis virus and application in situ hybridization

用限制酶HindⅢ和Ecorl双切鸡包涵体肝炎六邻体蛋白基因片段重组质粒 ,得到大小 6 36bp的基因片段 ,用地高辛标记制备DNA探针 ,其灵敏度为 0 .1～ 0 .3ng/ μL。用该探针对感染鸡包涵体肝炎病毒的雏鸡肝组织进行原位杂交 ,结果表明病毒经口感染后 12h肝细胞内出现病毒的复制 ,3～ 7d时病毒复制明显 ,9～ 16d病毒复制减弱。原位杂交表明鸡包涵体肝炎病毒主要定位于核内 ,同时也可进入胞浆中。地高辛标记鸡包涵体肝炎病毒DNA探针对检测该病毒DNA的灵敏度高 ,特异性强 ,操作方便 ,该探针可用于本病的分子病理学研究和特异性诊断。

The recombinant plasmids with the hexon gene fragment of fowl adenovirus Hb strain (FAV-Hb) were digested with restriction enzyme HindⅢ and Ecorl for preparation of the DNA probe.The obtained DNA forgments were further purified by electrop horesis,then the nucleotide sequence was analyzed.The results showed that the length of this DNA forgment was 636 bp.It was labeled by digoxigenin(DIG),and the efficient quantity of DIG labeled DNA was 0.1-0.3ng/ul.The lives of chickens infected with FAV-Hb were detected in situ hybridzation by this DNA probe.The positive cells64 were observed at 12 hours after infection (AI),and increased obviously at 3～7 days AI,and begun to decrease at 9～16 days AI.They were mainly in the nuclei siometimes,existed also in plasma.This DNA probe was sensitive,specific and convenient to detect the chicken inclusion boby hepatitis(IBH) virus,and can be used for the special diagnosis of IBH.