Method for improving immune protective rate of fasciola hepatica Cat L1 (FhCat L1) DNA (Deoxyribose Nucleic Acid) vaccine

A technology of immune protection rate and DNA vaccine, applied in recombinant DNA technology, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of insufficient immune protection rate, etc. The effect of rendering efficiency

Inactive Publication Date: 2013-05-08
SOUTHWEST UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, such an immune protection rate is still insufficient to develop a highly effective vaccine against Fasciola hepatica

Method used

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  • Method for improving immune protective rate of fasciola hepatica Cat L1 (FhCat L1) DNA (Deoxyribose Nucleic Acid) vaccine
  • Method for improving immune protective rate of fasciola hepatica Cat L1 (FhCat L1) DNA (Deoxyribose Nucleic Acid) vaccine
  • Method for improving immune protective rate of fasciola hepatica Cat L1 (FhCat L1) DNA (Deoxyribose Nucleic Acid) vaccine

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Experimental program
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Effect test

preparation example Construction

[0042] 3. Preparation of Cat L1 polyclonal antibody

[0043] Five 6-7-week-old female SD rats were intramuscularly injected with 30 μg / mouse of purified Cat L1 protein in the hind legs, and immunized for a total of 3 times, with an interval of 1 week between each time. Blood was collected 1 week after the last immunization to separate serum spare. Antibody concentration was determined by Coomasie protein assay.

[0044] 4. Identification of target protein by Western blotting

[0045] The protein purified by the His-Tag Fusion Protein Purification Kit in step 2 was subjected to SDS-PAGE (30V, 1h) electrophoresis and then transferred to a nylon membrane for Western blotting identification. Wash the membrane twice with 15ml 1×TBS, then add 5% non-fat milk powder TBS solution to block unbound protein sites. After washing twice with 1×TTBS, incubate the nylon membrane in polyclonal antibody for 1 hour, use the obtained Cat L1 polyclonal serum (diluted at 1:300), wash with 1×TT...

experiment example

[0066] The present invention is mainly divided into two major plates. One is to design and construct the plasmids required for the experiment, and the other is to evaluate the effect of the vaccine on rats. Elaborate in detail below in conjunction with accompanying drawing:

[0067] 1. Design and construct the plasmids required for the experiment

[0068] First, in order to obtain the candidate vaccine gene FhCat L1, fresh worms obtained from bovine liver were used to extract mRNA (see QIAGEN mRNA kit for specific extraction methods). Then obtain the cDNA of FhCat L1 gene and the required ORF fragment with RT-PCR method, and connect it in the pMD-18 cloning vector, form pMD-Cat L1 plasmid, then carry out PCR sequencing identification (see Figure 4 ), the sequencing results are shown in Figure 7 (SEQ ID NO.2), completely consistent with the expected result.

[0069] The next step is to express the FhCat L1 ORF fragment. At this time, the designed primers (Pe1, Pe2) can be...

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Abstract

The invention provides a method for improving immune protective rate of a fasciola hepatica Cat L1 (FhCat L1) DNA (Deoxyribose Nucleic Acid) vaccine. The method is carried out based on the presenting effect of the antigen in dendritic cells, the influential role mechanism that the single-chain antibody scFvNLDC-145 of the DEC-205 of surface molecules of dendritic cells (DC) can target the DC cells is utilized, so that the resistance of a Spragae-Dawley rat to fasciola hepatica infection subjected to the immune encoding of the DNA vaccine of the FhCat L1 is improved, and as a result, the immune protective rate of the DNA vaccine of the FhCat L1 is improved.

Description

technical field [0001] The invention relates to a method for improving the immune protection rate of the FhCat L1 DNA vaccine against Fasciola hepatica. Specifically related to single-chain antibody scFv utilizing dendritic cell (DC) surface molecule DEC-205 NLDC-145 Target DC cells to improve the resistance of Spragae-Dawley rats to Fasciola hepatica infection after immunization with DNA vaccine encoding FhCat L1 gene, thereby increasing the immune protection rate of anti-Fahsia hepatica Cat L1 DNA vaccine. Background technique [0002] Fasciola hepatica is a parasitic worm that can cause severe trematodesis in ruminants such as cattle and sheep as well as humans [1]. Worldwide, fascioliasis directly affects the development of animal husbandry and human health. It is estimated that it can cause economic losses of about 2 billion US dollars per year [2]. It is an important zoonotic parasitic disease that cannot be ignored. The prevention and treatment of this disease gener...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/00A61K47/42C12N15/12C12N15/79A61P33/12
Inventor 罗洪林
Owner SOUTHWEST UNIVERSITY
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