Expression of Sheep Interferon Alpha in Silkworm and Determination of Its Activity Against Peste Des Petits Ruminants Virus

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

Abstract

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

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.

Figures/Tables 6

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

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

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

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	-	-	-	-	-	+	+	+	-
	-	-	-	-	+	+	+	+	-
	-	-	-	+	-	-	+	+	-
	-	-	-	-	+	+	+	+	-
	-	-	±	+	+	+	±	+	-
	-	-	-	-	+	+	+	+	-
	-	-	-	-	+	+	+	+	-
	-	-	-	-	-	+	+	+	-

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

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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