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Preparation method and application of attenuated strain of contagious pustular dermatitis virus

A impetigo virus and infectious technology, which is applied in the field of preparation of double-gene deletion strains of sheep infectious impetigo virus, can solve the problems of unsatisfactory effectiveness and achieve the best immune protection and reduced pathogenicity

Pending Publication Date: 2022-04-15
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the lack of sheep infectious impetigo vaccine and the unsatisfactory effectiveness of known vaccines, and provides a construction of a fluorescence-labeled ovine infectious impetigo virus (ORFV) ORFs 120 / 121 double-gene deletion strain Method, the ORFs120 / 121 gene deletion strain (ORFV-SY17ΔORFs 120 / 121-eGFP) is to use homologous recombination technology to knock out the ORFs 120 / 121 gene from the ORFV-SY17 wild strain, and use green fluorescent protein (eGFP) gene is used as a screening marker to achieve further attenuation and reduce biosafety risks

Method used

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  • Preparation method and application of attenuated strain of contagious pustular dermatitis virus
  • Preparation method and application of attenuated strain of contagious pustular dermatitis virus
  • Preparation method and application of attenuated strain of contagious pustular dermatitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction, screening and identification of ovine infectious impetigo virus ORFs 120 / 121 double gene deletion strain

[0050] 1.1 Construction of recombinant shuttle plasmid pUC57-LFΔORFs 120 / 121-eGFP-RFΔORFs 120 / 121

[0051] According to the principle of optimal homology arm gene fragments, the upper and lower parts of the ORFV-SY17ORFs 120 / 121 gene sequence were selected as the recombined left and right homology arms. The sequence of the left homology arm (LF) is shown in SEQ ID NO.2, and the right homology The arm sequence (RF) is shown in SEQID NO.3. Further design and synthesis of specific primers for the left and right homology arms of the ORFV ORFs 120 / 121 gene, the primer sequences are shown in SEQ ID NO.4-7, respectively LFΔORFs 120 / 121-Fw: SEQ ID NO.4; LFΔORFs120 / 121 -Rv: SEQ ID NO.5; and RFΔORFs 120 / 121-Fw: SEQ ID NO.6; RFΔORFs 120 / 121-Rv: SEQ ID NO.7. Use PCR to amplify the sequences on both sides of the ORF120 / 121 gene of ORFV-SY17 strain as t...

Embodiment 2

[0061] Example 2 Toxicity and growth curve determination of ORFV-SY17ΔORFs 120 / 121-eGFP double gene deletion attenuated strain

[0062] After repeatedly freezing and thawing ORFV-SY17ΔORFs 120 / 121-eGFP double gene deletion attenuated strain was diluted 10 times in DMEM (10 -1 -10 -10 ), inoculated in a 96-well cell culture plate with cells growing to 80% monolayer, inoculated 1 longitudinal row (8 wells) for each dilution, 100 μL in each well; at the same time set normal cell control, observed and recorded the lesions of each dilution every day The number of wells, the virus titer of the ORFV-SY17ΔORFs120 / 121-eGFP double gene deletion attenuated strain was determined according to the Karber method, TCID 50 for 10 5.5 / 0.1mL, indicating that the deletion of ORFs 120-121 gene has little effect on its replication in OFTu cells. Afterwards, single-gene deletion strain ORFV-SY17ΔORF 120-eGFP, double-gene deletion strain ORFV-SY17ΔORFs 120 / 121-eGFP, wild strain ORFV-SY17 were tes...

Embodiment 3

[0063] Example 3 Evaluation of safety and immune protection effect of ORFV ORFs120 / 121 double-gene deletion attenuated strain

[0064] Nine 3-6-month-old lambs with negative ORFV antibody tests were randomly divided into 3 groups, and the inoculation method was inoculated with ORFV-SY17 (n=3) by scratching the lower lip. ORFV-SY17ΔORFs 120 / 121-eGFP Virus strain (n=3), DMEM liquid (n=3), inoculum dose is 10 6.5 TCID 50 , Observe and record whether the characteristic "mouth sore" lesions appear on the lips of the inoculated lambs every day. The results showed that the ORFV-SY17 wild strain could observe lesions on the 3rd day after infection, and the pustules and scabs were extremely obvious on the 5th to 7th day. Figure 7 h-k; As the time prolongs for 5-7 days, whether ORFV-SY17ΔORFs 120-eGFP single gene deletion attenuated strain group or ORFV-SY17ΔORFs 120 / 121-eGFP double gene deletion attenuated strain group, compared with ORFV-SY17 wild strain group All improved. Howev...

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Abstract

The invention provides a preparation method of a contagious pustular dermatitis virus attenuated strain, which is characterized in that virulence genes of contagious pustular dermatitis virus are mutated and inactivated, and the virulence genes are mutated into ORFs 120 / 121 double-gene mutation. After the ORFs 120 / 121 gene is deleted, the pathogenicity of the ORFV is obviously reduced. According to the present invention, the ORFs 120 / 121 gene of the ORFV-SY17 strain is knocked out to obtain the double-gene-deleted contagious pustular dermatitis virus attenuated strain, and the double-gene-deleted contagious pustular dermatitis virus attenuated strain has potential application value in preparation of the contagious pustular dermatitis gene-deleted attenuated live vaccine production strain. The deleted ORFs 120 / 121 gene can also be used as a marker gene to establish a differential diagnosis method for ORFV vaccine virus and wild virus detection.

Description

technical field [0001] The invention relates to the field of veterinary medicine, in particular to a method for preparing a fluorescence-labeled double-gene deletion strain of sheep infectious impetigo virus and its application. Background technique [0002] Ovine impetigo, commonly known as "Orf", is an acute, highly contagious zoonotic disease of sheep, goats, etc. caused by Orf virus (ORFV) infection. . The disease is a common disease in sheep flocks, mainly harming 3-6 month-old lambs, resulting in the decline of their growth / reproductive performance and even death, causing serious economic losses to the sheep industry in my country and the world. Due to the strong viability of ORFV in vitro, it is difficult to clear the flock once infected, and it can continue to cause harm to the flock for many years. Therefore, the disease is not only a serious hazard to the sheep farming industry, but also practitioners engaged in breeding, slaughtering, veterinary and other occupa...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/39C12N15/85A61K39/275A61P31/20C12Q1/70C12N15/11C12R1/93
Inventor 赵魁王帅何师师方梓煜周艳龙关继羽陆慧君吕品徐梦实贺文琦高丰
Owner JILIN UNIV
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