羊α干扰素在家蚕中的表达及抗小反刍兽疫病毒活性测定

doi:10.13560/j.cnki.biotech.bull.1985.2021-0195

摘要/Abstract

摘要：

干扰素具有广谱抗病毒、抗肿瘤活性,可增强机体抗病毒能力,已经广泛应用于病毒性疾病的防控。小反刍兽疫病毒是危害山羊、绵羊等小反刍动物常见的病毒之一,该病毒的传播对全球养殖业造成了严重的影响。为了表达具有高效抗病毒活性的羊α干扰素（OviIFN-α）,将OviIFN-α基因序列根据家蚕密码子偏好性进行优化合成,构建pVL1393-OviIFN-α转移载体,将转移载体与ORF1629缺失型杆状病毒基因组DNA利用脂质体介导的转染法在BmN细胞中进行重组,重组病毒感染家蚕,在蚕血淋巴液中获取重组蛋白OviIFN-α。利用微量细胞病变抑制法在BHK-VSV*GFP系统中检测到该蛋白表达量可达6.5×10⁵U/mL左右。小反刍兽疫病毒增殖抑制实验显示,羊α干扰素对小反刍兽疫病毒在BHK细胞中的复制产生了明显的抑制作用,IC₅₀为136.93 U左右。该结果为羊α干扰素用于小反刍兽疫疫情防控提供了重要的理论依据。

关键词: 羊α干扰素, 杆状病毒表达系统, 小反刍兽疫病毒, 抗病毒活性

Abstract:

Interferon has broad-spectrum anti-viral and anti-tumor activities and can enhance the body’s anti-viral ability,thus interferon has been widely used in the prevention and control of viral diseases. Peste des petits ruminants virus（PPRV）is one of the common viruses that harm small ruminants such as goats and sheep. The spread of this virus has resulted in a serious impact on the global breeding industry. In order to express sheep interferon alpha（OviIFN-α）with high antiviral activity,the OviIFN-α gene sequence was optimized and synthesized according to the codon preference of the silkworm to construct pVL1393-OviIFN-α transfer vector,and then the transfer vector and baculovirus genomic DNA with deleted of ORF1629 sequence was recombined in BmN cells by liposome-mediated transfection. The recombinant virus infected the silkworm,and the recombinant protein OviIFN-α was obtained from the silkworm hemolymph. The antiviral titer of the protein detected in the BHK-VSV*GFP system using the cytopathic inhibition method reached about 6.5×10⁵ U/mL. In addition,the experiments on the proliferation inhibition of PPRV showed that OviIFN-α significantly inhibited the replication of PPRV in the BHK cells,with an IC₅₀of about 136.93 U. This result provided an important theoretical basis for OviIFN-α to be used in the prevention and control of peste des petits ruminants disease.

Key words: sheep interferon alpha, baculovirus expression system, peste des petits ruminants virus, antiviral activity

李丹, 杜梦潭, 修明霞, 刘兴健, 张志芳, 李轶女. 羊α干扰素在家蚕中的表达及抗小反刍兽疫病毒活性测定[J]. 生物技术通报, 2022, 38(1): 187-193.

LI Dan, DU Meng-tan, XIU Ming-xia, LIU Xing-jian, ZHANG Zhi-fang, LI Yi-nv. Expression of Sheep Interferon Alpha in Silkworm and Determination of Its Activity Against Peste Des Petits Ruminants Virus[J]. Biotechnology Bulletin, 2022, 38(1): 187-193.

图/表 6

图1 重组质粒的酶切鉴定 M：Marker;1：pVL1393-OviIFN-α的BamH Ⅰ、EcoR Ⅰ双酶切产物

Fig. 1 Enzyme digestion identification of recombinant plasmid M：Marker. 1：BamH I and EcoR I double enzyme digestion products of pVL1393-OviIFN-α

图2 重组病毒感染家蚕后蚕血淋巴中目的基因的PCR鉴定 M：Marker;1：以OviIFN-α样品DNA为模板的PCR产物;2：以对照蚕血DNA为模板的PCR产物

Fig.2 PCR identification of the target gene in the hemoly-mph of the silkworm infected with the recombinant virus M：Marker. 1：PCR product using OviIFN-α sample DNA as template. 2：PCR product using control silkworm blood DNA as template

图3 不同浓度干扰素处理组后BHK细胞感染VSV增殖情况 A：高浓度干扰素抑制VSV复制;B：低浓度干扰素处理部分抑制病毒复制;C：VSV病毒对照组;D：空白细胞对照组

Fig. 3 Proliferation of BHK cells infected with VSV after treatment with different concentrations of interferon A：High-concentration interferon inhibits VSV replication. B：Low-concentration interferon treatment partially inhibits virus replication. C：VSV virus control group. D：Blank cell control group

表1 重组羊α干扰素在BHK细胞中抑制VSV*GFP病毒活性检测

Table 1 Recombinant goat interferon alpha inhibits VSV*GFP virus activity in BHK cells

干扰素 Interferon	蚕血淋巴液稀释倍数 Silkworm hemolymph dilution factor							病毒对照Virus control	空细胞对照 Blank cell control
干扰素 Interferon	5.0×10³	1.0×10⁴	2.0×10⁴	4.0×10⁴	8.0×10⁴	1.6×10⁵	3.2×10⁵	病毒对照Virus control	空细胞对照 Blank cell control
OviIFN-α	-	-	-	-	-	+	+	+	-
	-	-	-	-	+	+	+	+	-
	-	-	-	+	-	-	+	+	-
	-	-	-	-	+	+	+	+	-
	-	-	±	+	+	+	±	+	-
	-	-	-	-	+	+	+	+	-
	-	-	-	-	+	+	+	+	-
	-	-	-	-	-	+	+	+	-

表2 PPRV的TCID50测定结果

Table 2 TCID50 measurement results of PPRV

病毒稀释度Virus dilution	细胞培养孔数 Number of cell culture holes		累计孔数 Cumulative hole number		比值 Ratio	百分数 Percentage
病毒稀释度Virus dilution	CPE	No CPE	CPE	No CPE	比值 Ratio	百分数 Percentage
10^-1	8	0	36	0	36/36	100
10^-2	8	0	28	0	28/28	100
10^-3	8	0	20	0	20/20	100
10^-4	8	0	12	0	12/12	100
10^-5	4	4	4	4	1/2	50
10^-6	0	8	0	12	0/12	0

图4 羊α干扰素抑制BHK细胞感染PPRV增殖效果图 A：PPRV感染组;B：羊α干扰素抑制PPRV感染BHK细胞,未出现合胞体;C：空白细胞对照组

Fig. 4 Effects of OviIFNα on inhibiting the proliferation of BHK cells infected with PPRV A：PPRV infection group. B：OviIFNα inhibits PPRV infection of BHK cells without CPE. C：Blank cell control group

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