重组AcMNPV感染家蚕后造成宿主组织液化的机制分析

杆状病毒的组织蛋白酶(V-CATH)和几丁质酶(V-CHIA)是病毒感染宿主后造成宿主组织液化的关键酶。家蚕核型多角体病毒(BmNPV)感染家蚕后会导致宿主组织的液化,而苜蓿银蚊夜蛾核型多角体病毒(AcMNPV)感染家蚕后不会造成宿主组织的液化。为了分析AcMNPV不造成家蚕宿主组织液化的原因,利用Bac-to-Bac系统将BmNPV的v-cath(Bmcath)、v-chiA(BmchiA)同时或分别重组到AcMNPV的极晚期强启动子p10和polh下游,并以同样的方法将AcMNPV的v-cath(Accath)、v-chiA(AcchiA)同时或分别重组到AcMNPV的p10和polh下游,从而构建6种重组AcMNPV。结果显示6种重组AcMNPV均能感染家蚕,并造成家蚕宿主组织的液化,说明重组AcMNPV中v-cath和v-chiA的过量表达在病毒对家蚕组织液化过程中发挥了重要作用。通过对感染6种重组AcMNPV之间的蚕体液化发生时间和液化率进行比较,发现相对于v-chiA,v-cath对组织液化的作用可能更加直接。检测比较感染AcMNPV和重组AcMNPV家蚕血淋巴中的组织蛋白酶和几丁质酶活性,两种酶的活性均随v-cath和v-chiA基因拷贝数的增加而增加。AcMNPV感染后不会造成家蚕宿主组织液化可能是由于病毒的组织蛋白酶活性太低,而不是由Bmcath和Accath、BmchiA和AcchiA之间的序列差异造成的。

An Analysis on Liquefaction Mechanism of Silkworm Tissues After Infection by Recombinant AcMNPV

Baculovirus-encoded cathepsin(V-CATH) and chitinase(V-CHIA) are two key enzymes involved in the liquefaction of host insect tissues after infection by baculovirus.Liquefaction occurred in tissues of silkworm(Bombyx mori) larvae after infection by B.mori nucleopolyhedrovirus(BmNPV),but not in tissues of silkworm larvae infected by Autographa california multiple nucleopolyhedrovirus(AcMNPV).In order to analyze reasons why AcMNPV does not cause liquefaction in host silkworm tissues,BmNPV v-cath(Bmcath) and v-chiA(BmchiA) were cloned simultaneously or separately into downstream of the very-late strong promoter p10 and polh of AcMNPV using Bac-to-Bac baculovirus expression system,and AcMNPV v-cath(Accath) and v-chiA(AcchiA) were cloned simultaneously or separately into the downstream of p10 and polh of AcMNPV using the same approach to construct 6 kinds of recombinant AcMNPV.The results showed that all 6 kinds of recombinant AcMNPV could infect silkworm and cause liquefaction of host silkworm tissues,suggesting that the over-expression of v-cath and v-chiA of recombinant AcMNPV played an important role in the liquefaction of silkworm larval tissues.Through comparing liquefaction occurrence time of silkworm tissues and liquefaction rate between 6 kinds of recombinant AcMNPV,it was found that the effect of v-cath to tissue liquefaction was more direct compared to that of v-chiA.Determination of cathepsin and chitinase activities in the hemolymph of AcMNPV-and recombinant AcMNPV-infected silkworm larvae indicated that the activity of cathepsin and chitinase increased with the copy number of v-cath and v-chiA genes,respectively.It is thus suggested that non-liquefaction of silkworm larvae infected by AcMNPV is due to low cathepsin activity rather than sequence difference between Bmcath and Accath,and between BmchiA and AcchiA.