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Recombined subunit vaccine of haemaphysalis concinna and preparation method thereof

A subunit vaccine, the technology of the blood tick, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of pollution, drug residue environment, etc.

Inactive Publication Date: 2010-06-30
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art, that is, to overcome the residue of drugs in animal products and the problems of environmental pollution, and finally to develop a recombinant subunit vaccine of Haemophilus swarmophilus with good immunogenicity

Method used

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  • Recombined subunit vaccine of haemaphysalis concinna and preparation method thereof
  • Recombined subunit vaccine of haemaphysalis concinna and preparation method thereof
  • Recombined subunit vaccine of haemaphysalis concinna and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Acquisition of the Hc-23 Antigen Gene Fragment of Haemophilus swarmophilus

[0032] A pair of specific primers were designed and synthesized using the known nucleotide sequences of the protective antigen gene P27 / 30 of the Chinese strain and the Japanese strain of Haemophilus longhorn. The primer is used to carry out RT-PCR on the total RNA of the haemophilus tick, and the cDNA generated by reverse transcription is used as a template, and the specific primer of the gene is used to amplify to obtain the target fragment.

[0033] The experimental haemophilus ticks were collected from rabbits in Cangxi County, Sichuan Province. They were identified as haemophilus haemophilus by morphological identification. They were used as experimental ticks after four generations of rabbit breeding under laboratory conditions. The experimental ticks in the non-parasitic stage were all in Cultivated in an incubator under laboratory conditions (Deng 1978).

[0034]As experiment...

Embodiment 2

[0036] Preparation of Example 2 Haemophilus subunit vaccine

[0037] Design a pair of prokaryotic expression primers according to the obtained Hc-23 gene sequence, and design the primers containing EcoR I and HindIII restriction sites respectively as follows:

[0038] 5'-CG GAATTC ATGGGAGACGAGGAGAAGAG-3';

[0039] 5'-GGG TTCGAA TTGCCTCGTTGTTTTATTCCT-3'

[0040] Use the Hc-23 gene as a template for PCR amplification, and double-digest the recovered PCR product with a restriction endonuclease, then connect it with the expression vector (pMD18-T) that has undergone the same digestion treatment, and transform it into the receptor Bacteria to obtain a recombinant expression vector (pMD18-T-Hc-23 plasmid) containing the Hc-23 gene;

[0041] The positive clone plasmid (pMD18-T-Hc-23 plasmid) with correct sequencing was used as a template for PCR amplification, and the target fragment was recovered for subcloning, that is, the PCR product was cloned into pMD18-T vector, and final...

Embodiment 3

[0047] Embodiment 3 Application effect test of the recombinant subunit vaccine of the present invention:

[0048] (1) Rabbit Anti-Hemophilus Tick Immune Serum Preparation

[0049] Artificially infect Chinese big-eared white rabbits with Haemophilus swarms, inoculate each ear with 30 adult ticks, 60 nymphs and 100 nymphs, and repeat once after the full blood falls, a total of two times, blood is collected to separate the serum, and the serum Store at -20°C for later use.

[0050] (2) Western blot

[0051] After the purified protein was subjected to SDS-PAGE, it was transferred to a nitrocellulose membrane, incubated with 1% BSA for 30 min, and then washed with 0.05% Tween-20 PBS-T, and the positive serum of rabbit anti-Hemophilus swarmophilus was added (according to 1:100 Diluted) as the primary antibody, reacted at 4°C for more than 12 hours, discarded the primary antibody, washed the membrane with 0.1M PBS-T, and added the secondary antibody goat anti-rabbit IgG-HRP (Chengd...

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Abstract

The invention discloses a recombined subunit vaccine of haemaphysalis concinna and a preparation method thereof. The recombined subunit vaccine is formed by mixing antigenic gene recombined protein of the haemaphysalis concinna and Freund's complete adjuvant (FCA), wherein the content of the antigenic gene recombined protein is 50 mg / ml, that is 50 mL rHc-23 and 950 ml Freund's complete adjuvant are mixed to prepare the recombined subunit vaccine of the haemaphysalis concinna; the amino acid sequence of the antigenic gene recombined protein is shown in the table SEQID NO:1; and the nucleotide sequence of the antigenic gene is shown in the table SEQID NO:2. Through screening and cloning, prokaryotic expression and separation and purification of protective antigen gene of the haemaphysalis concinna and the application effect test of the recombined subunit vaccine, the method shows that an expression product of recombined vector bacteria can be identified by rabbit anti-haemaphysalis concinna positive serum. In an animal immunity test, after rHc-23-FCA is subjected to three immune rabbits, the blood saturation rates of haemaphysalis larva, haemaphysalis middle and haemaphysalis imago are 40.3 percent, 45.6 percent and 41.3 percent respectively; and compared with the blood saturation rates of the haemaphysalis larva, haemaphysalis middle and haemaphysalis imago in a contrast set: 90.1 percent, 94.3 percent and 97.7 percent, the differences are remarkable. The method promotes the development of anti-haemaphysalis immunity, haemaphysalis control and haemaphysalis disease spread work.

Description

technical field [0001] The invention relates to a recombinant subunit vaccine, in particular to a recombinant subunit vaccine of Haemophilus swarmophilus and a preparation method thereof. Background technique [0002] Haemaphysalis concinna is an obligate blood-sucking insect on the surface of animals. It is widely distributed in various countries in the world. It has been found on various animals in more than 10 provinces and regions in my country. The haemophilus tick bites and sucks a large amount of blood from the host, causing local skin swelling, inflammation, itching and ulceration of the host [1] . In addition, this tick can also transmit a variety of pathogens, and is one of the important tick species that seriously endanger my country's animal husbandry[ [2-11] ]. [0003] The tick is known to be distributed in many countries and regions of the world [12-19] , have also been reported in many provinces in China [5，20-21] . Since the late 1990s, epidemics of ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P33/14C07K14/435C12N15/12C12N15/70
Inventor 杨光友边尧彭雪蓉古小兵王淑贤
Owner SICHUAN AGRI UNIV
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