Rabbit Pasteurella multocida DNA vaccine and preparation method

A DNA vaccine, Pasteurella rabbit technology, applied in the field of molecular biology and immunology, can solve the problems of complicated operation, poor stability of immune effect, etc., and achieve the effects of low preparation cost, convenient immunization operation, and good cellular immunity

Inactive Publication Date: 2016-05-04
SHANGHAI TRUELAND INFORMATION & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immune effect of rabbit Pasteurella inactivated vaccine or multiple va

Method used

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  • Rabbit Pasteurella multocida DNA vaccine and preparation method
  • Rabbit Pasteurella multocida DNA vaccine and preparation method
  • Rabbit Pasteurella multocida DNA vaccine and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, rabbit Pasteurella multocida OmpH 1 Genetic DNA Vaccine Preparation

[0025] 1. Design of expression primers

[0026] According to the genome sequence of Pasteurella rabbit, designed to amplify Pasteurella multocida (C51-2-499) OmpH 1 Gene two primers P1 and P2, and introduced BamHI and XhoI restriction sites at the two 5' ends respectively.

[0027] Primer sequence: P1: 5′T GGATCC ATGAAAAAGACAATCGTAC3' (SEQ ID NO: 2);

[0028] P2: 5′AT CTCGAG TGTACGCGTAAACCT3' (SEQ ID NO: 3).

[0029] 2. Cloning of the target gene

[0030] ① Extraction of total DNA of Pasteurella multocida in rabbits

[0031] Take 2mL of the bacterial solution in a microcentrifuge tube, and centrifuge at 6000r / min for 5min to collect the precipitate. Dissolve the precipitate with 500 μL NET buffer and place it in a water bath at 80°C for 15 minutes; take out the microcentrifuge tube from the water bath and place it at room temperature for 2 minutes, then add 12 μL lysozyme, and d...

Embodiment 2

[0042] Example 2, OmpH 1 Immunization of mice with genetic DNA vaccine elicits humoral immune response

[0043] Select 15 healthy female BALB / C mice of 6-8 weeks and divide them into 3 groups, 5 in each group, respectively: normal saline group; liposome-coated pET30-OmpH 1 Vaccine group; Liposome control group.

[0044] Liposome-coated OmpH 1 Preparation of DNA vaccine: Take 2 μg / μL of OmpH 1 After mixing 0.5mL of DNA vaccine with 20000.5mL of liposome, let it stand at room temperature for 20min, and the liposome-coated OmpH 1 DNA vaccine. OMP 1 The DNA content was 1 μg / μL, and the dose for each injection was 100 μL / mouse. The liposome control group was coated with liposome-coated OmpH 1 The same amount of liposomes of the DNA vaccine immunization group (without OmpH 1 DNA vaccine) to immunize mice. All mice were immunized once at intervals of 2 weeks, and immunized 3 times in total.

[0045] Experimental results: 7, 21, 35 days after immunization, the blood was coll...

Embodiment 3

[0050] Example 3, OmpH 1 Cellular Immune Response Elicited by Gene DNA Vaccine Immunization in Mice

[0051] 1. Lymphocyte proliferation test

[0052] Before the second immunization described in Example 2, before the third immunization and before the challenge, the mice were killed by decapitation, the spleen was aseptically collected to prepare a spleen cell suspension, and the cell concentration was adjusted to 1×10 7 individual / mL. 50 μL of splenocyte suspension was added to each well of a 96-well cell culture plate, and a negative control was set at the same time. The test wells and the negative control wells were set in triplicate respectively, and 50 μL of 20 mg / l extracted Pasteurella OmpH was added to each well of the test wells. 1 protein, add 50 μL of cell culture medium to each well of negative control wells, place at 37°C, 5% CO 2 After culturing in the incubator for 48h, add 5g / LMTT10μL to each well and place in the incubator for 3h. Then add 100μSDS-HCl to e...

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Abstract

The invention belongs to the field of molecular biology and immunology, and discloses a rabbit Pasteurella multocida DNA vaccine and a preparation method. The rabbit Pasteurella multocida DNA vaccine contains recombinant plasmid of a rabbit Pasteurella multocida Omp H1 gene. The DNA vaccine is inoculated through intramuscular injection, no subcutaneous injection is needed, and immunization is convenient. Meanwhile, the DNA vaccine will not cause enhancement of toxicity or gene integration, rabbit head tilt or tic or acute death or the like will not be caused, and the DNA vaccine is safe and free of side effect. An experiment shows that the rabbit Pasteurella multocida DNA vaccine can induce rabbits to generate good humoral immunity and cell immunity, and is applicable to immunization of rabbit Pasteurella multocida, and occurrence and epidemic of the rabbit Pasteurella multocida are prevented. The rabbit Pasteurella multocida preparation method is simple, low in preparation cost and suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of molecular biology and immunology, in particular to a DNA vaccine of Pasteurella multocida and a preparation method thereof, in particular to a DNA vaccine based on Pasteurella multocida OmpH 1 Genetic DNA vaccines. Background technique [0002] Pasteurellosis in rabbits is a zoonotic polymorphic infectious disease caused by Pasteurella multocida. When poor feeding management, nutritional deficiencies, sudden changes in feed, excessive fatigue, long-distance transportation, parasite infection, cold, stuffy heat, humidity, crowding, poor ventilation in pens, and continuous rain, etc., the rabbit's resistance is reduced, and germs are easy to take the opportunity to invade the body. , an endogenous infection occurs. The feces and secretions of sick rabbits can continuously discharge virulent germs, pollute feed, drinking water, utensils and the external environment, and infect healthy rabbits through the digestive t...

Claims

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Application Information

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IPC IPC(8): A61K39/102A61P31/04C12N15/85
CPCA61K9/127A61K39/102A61K2039/54A61K2039/545A61K2039/552A61K2039/575C07K14/285C12N15/85C12N2800/107
Inventor 王开
Owner SHANGHAI TRUELAND INFORMATION & TECH CO LTD
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