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对虾白斑综合症病毒(WSSV)的致病性强、危害性大、地域分布和宿主范围广泛,目前还不能有效地控制疫情。将含有WSSV囊膜蛋白Vp28基因的重组杆状病毒HyNPV-Vp28感染家蚕(Bombyx mori)蛹,对发病蚕血淋巴进行SDS-PAGE和Western blotting分析,结果表明Vp28在家蚕体内得到了表达。将重组病毒囊膜蛋白rVp28疫苗配制成药饵,持续口服免疫75天,对克氏原螯虾进行预防WSSV,实验虾分为2%重组Vp28疫苗、2%普通蚕蛹组织匀浆(阳性对照)和普通饵料(阴性对照)3个处理组。免疫35天后进行口服攻毒,20天内rVp28疫苗组的累积存活率为63.33%,与阳性和阴性对照比差异显著(P<0.05),PRP分别达54.16%和59.26%;注射攻毒后20 天内rVp28疫苗组的累积存活率与阳性和阴性对照组比差异不显著(P>0.05),PRP分别为46.12% 和49.99%。第55天对存活虾再口服攻毒,20天内rVp28疫苗组与阳性和阴性对照组比累积存活率差异显著(P<0.05),PRP分别为55.80%和63.16%;二次注射攻毒后,rVp28疫苗组的PRP均为31.25%。对vVp28疫苗组存活虾的胃、肠和肝胰腺组织进行病毒的原位杂交检测均呈阴性反应,而对照组死亡虾组织都呈阳性反应。本研究表明,口服免疫家蚕蛹表达的病毒囊膜蛋白Vp28能诱导螯虾产生抗病毒保护作用,对应用疫苗预防对虾的病毒性疾病具有重要意义。
White spot syndrome virus (WSSV) is highly pathogenic, highly harmful and has a wide geographical distribution and host range. Currently, the epidemic situation can not be effectively controlled. The recombinant baculovirus HyNPV-Vp28 containing the Vp28 gene of WSSV was infected into the pupa of Bombyx mori. SDS-PAGE and Western blotting analysis of the silkworm hemolymph showed that Vp28 was expressed in silkworm. The recombinant viral envelope protein rVp28 vaccine formulated as a bait, continuous oral immunization for 75 days, the Procambarus clarkii prevention WSSV, experimental shrimp is divided into 2% recombinant Vp28 vaccine, 2% ordinary silkworm pupa homogenate (positive control) and ordinary Feed (negative control) 3 treatment groups. The cumulative survival rate of rVp28 vaccine group was 63.33% within 20 days after oral immunization. The difference between the two groups was significant (P <0.05), and the PRP reached 54.16% and 59.26%, respectively. Within 20 days after injection The cumulative survival rate of the rVp28 vaccine group was not significantly different from that of the positive and negative controls (P> 0.05). The PRP was 46.12% and 49.99% respectively. On the 55th day, the survival shrimp were re-challenged with the virus. The cumulative survival rate of the rVp28 vaccine group was significantly higher than that of the positive and negative control groups within 20 days (P <0.05), and the PRP was 55.80% and 63.16% respectively. After the secondary injection, The PRP of the rVp28 vaccine group was 31.25%. In situ hybridization detection of virus in the stomach, intestine and hepatopancreas of survivor shrimp in the vVp28 vaccine group showed negative reaction, while the control group died shrimp tissue were positive. This study showed that the oral immunization of silkworm pupa expression of viral envelope protein Vp28 can induce the anti-virus protection of crayfish, the vaccine for the prevention of shrimp virus disease is of great significance.