Foot and mouth disease virus inhibitor and preparation method and application thereof

A foot-and-mouth disease virus and anti-foot-and-mouth disease virus technology, which is applied in the field of genetic engineering, can solve problems such as difficulties in the prevention and control of foot-and-mouth disease, and achieve the effects of improving resistance and being convenient to use.

Inactive Publication Date: 2010-06-16
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In nature, there are many mutant strains of various viruses, and the foot-and-mouth disease virus has seven serotyp

Method used

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  • Foot and mouth disease virus inhibitor and preparation method and application thereof
  • Foot and mouth disease virus inhibitor and preparation method and application thereof
  • Foot and mouth disease virus inhibitor and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] U6 promoter of ribosomal RNA was amplified from mouse NIH / 3T3 cells by PCR. The promoter before the multiple cloning site of plasmid pcDNA3.1 was replaced with this promoter sequence.

[0039] Chemically synthesize each sequence of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO5, the inverted repeat sequence of each sequence and the DNA sequence of the hairpin linker. Each sequence was ligated with the corresponding inverted repeat sequence through a hairpin linker DNA to form the siRNA template sequence. Each siRNA template sequence was inserted behind the U6 promoter to construct p3D-NT56, p3D1, p1A-NT65, p1A-NT19 and p2B, respectively. The inhibition of these plasmids against different serotypes of foot-and-mouth disease virus strains was tested at the level of cells and suckling mice. Then the plasmid p3D-NT56 with cross-protection for each strain was treated with CaCl 2 Transformation into intermediate host attenuated Salmonella typhimurium LB50...

Embodiment 2

[0042] With the recombinant live bacteria inhibitor that the present invention forms with 10 7 ~10 10 The dose of CFU / only is carried out to the guinea pig that body weight is 250-300 gram to carry out hind leg vastus medial muscle injection (use plate dilution counting method to calculate the number of viable bacteria). Use 50ID after 36 hours 50 of foot-and-mouth disease virus in guinea pigs subjected to a hind paw puncture challenge. The results showed that the guinea pigs that had not been injected with live bacteria inhibitors were all sick within 48 hours of the FMD virus challenge, while the guinea pigs that had been injected with live bacteria inhibitors had strong resistance to the challenge of FMD virus, about 80% of the guinea pigs Finally, it was protected (Table 3), and the diseased guinea pigs injected with the live bacteria inhibitor were much milder in symptoms than the control animals.

Embodiment 3

[0044] With the recombinant live bacteria inhibitor that the present invention forms with 10 8 ~10 11 The dose of CFU / only is injected intramuscularly into the neck of domestic pigs. Use 50ID after 24 hours 50 Neck intramuscular challenge of domestic pigs with foot-and-mouth disease virus. Seven days and fourteen days after the challenge, blood was collected from domestic pigs, the blood was allowed to stand overnight, centrifuged at 3500r / min for 10min, and the supernatant serum was taken. Detection of neutralizing antibodies and nonstructural protein antibodies. The results showed that domestic pigs that had not been injected with inhibitors of live bacteria all fell ill within 4 days of FMD virus challenge, while pigs that had been injected with inhibitors of live bacteria in advance were protected by 100% to 10 days ( figure 1 ). The results of serological analysis showed (Table 4) that the titers of neutralizing antibodies and non-structural protein antibodies in una...

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Abstract

The invention belongs to the genetic engineering field, in particular relates to a foot and mouth disease virus inhibitor which is designed by adopting RNAi technology and a preparation method and application thereof. The inhibitor is a recombinant live bacterial vaccine containing plasmid for expressing siRNA of foot and mouth disease virus and uses attenuated salmonella suipestifer as recipientbacterium. The recombinant live bacterial vaccine of the invention is convenient to store and use. The inhibitor can be given by injection as well as by mouth. The attenuated salmonella is used as carrier to transfer the expression plasmid of siRNA so that the organism can resist the targeting virus of siRNA and can also resist the infection of salmonella.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a foot-and-mouth disease virus inhibitor designed by using RNAi technology, a preparation method and application thereof. Background technique [0002] Foot and mouth disease (FMD) is the most serious livestock infectious disease in the world today, mainly harming pigs, cattle, sheep and other cloven-hoofed animals. For many years, foot-and-mouth disease has broken out and become popular in a large scale all over the world, causing huge economic losses to animal husbandry. According to immunogenicity, there are seven serotypes of FMD virus: A, O, C, SATI, SATII, SATIII and AsiaI. The inactivated vaccines currently on the market have immune protection for livestock, but it takes about 10 days after the vaccine is injected to induce a large amount of antibodies in the body to protect the animals. Foot-and-mouth disease virus can cause a large number of animals to be...

Claims

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Application Information

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IPC IPC(8): A61K39/135A61K39/295A61P31/14A61K39/112
CPCY02A50/30
Inventor 刘明秋严维耀丛薇金虹郑兆鑫
Owner FUDAN UNIV
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