Bivalent egg yolk antibody against DVH (duck virus hepatitis) as well as preparation method and application of bivalent egg yolk antibody

A duck viral hepatitis, egg yolk antibody technology, applied in the direction of virus/phage, antiviral immunoglobulin, botanical equipment and methods, etc., can solve the problems of lack of effective control measures and lack of cross protection of DVH, and achieve prevention and control. Duck viral hepatitis, significant social benefit, stable use effect

Active Publication Date: 2017-06-16
PU LIKE BIO ENG
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The research results have shown that DHAV-1 and DHAV-3 are prevalent in many areas of my country at the same time, and there is a lack of cross protection between the two, and there is still a lack of effective control measures for DVH caused by DHAV-3

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  • Bivalent egg yolk antibody against DVH (duck virus hepatitis) as well as preparation method and application of bivalent egg yolk antibody
  • Bivalent egg yolk antibody against DVH (duck virus hepatitis) as well as preparation method and application of bivalent egg yolk antibody
  • Bivalent egg yolk antibody against DVH (duck virus hepatitis) as well as preparation method and application of bivalent egg yolk antibody

Examples

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preparation example Construction

[0042] The preparation method of the duck viral hepatitis bivalent egg yolk antibody adopts the following steps: breeding of duck hepatitis virus DHAV-1 and DHAV-3 seed virus, preparation of vaccine for immunization, immunization of laying hens, and extraction of egg yolk antibody. The selected DHAV-1 strain in the embodiment of the present invention is DRL-62 strain, and DHAV-3 strain is SD1 strain, and its specific method is:

[0043] 1. Source of virus species Duck hepatitis virus DRL-62 strain (preserved by ATCC, deposit number VR-1313); SD1 strain was isolated from Pulaike Bioengineering Co., Ltd. (CCTCC deposit, deposit number: CCTCC NO.V201225).

[0044] 2. Propagation of virus seeds Dilute duck hepatitis virus DRL-62 strain virus seeds with sterilized PBS solution at 1:100, inoculate 9-11-day-old SPF chicken embryos through the allantoic cavity, 0.1ml per embryo. Continue incubation at 37°C, discard dead embryos within 48 hours, harvest dead chicken embryos within 48-9...

Embodiment 1

[0062] Example 1 Molecular biological identification of DHV SD1 isolates

[0063] 1. Duck hepatitis virus SD1 strain virus is a newly isolated and identified virus, which has the following characteristics:

[0064] 1.1 The clinical symptoms caused by DHAV-3 and DHAV-1 are very similar. The main manifestations are: sudden onset of ducklings, obvious neurological symptoms, convulsions, and death soon. After death, ducklings are in opisthotonus posture. The pathological changes showed that the liver was enlarged, and there were a large number of bleeding points and spots on the liver surface, and the bleeding became more obvious with the prolongation of the death time.

[0065] 1.2 According to the DHV genome sequence published in GenBank, several pairs of specific primers were designed, the VP1 gene was amplified by conventional RT-PCR method, and the sequence analysis of the VP1 gene of the isolate SD1 was carried out. The results showed that the VP1 gene of SD1 had the highes...

Embodiment 2

[0097] Embodiment 2 Serum cross-neutralization test

[0098] The serum was fixed and diluted, and the positive serum of the DHV DRL-62 strain and the SD1 isolate were serially diluted 2 times with normal saline. Take 1.0ml of the virus solution of DRL-62 strain and SD1 isolate containing 200ELD50 / 0.2ml and mix them with equal amounts of positive sera of different dilutions of each strain, and act at 37°C for 1h. At the same time, virus and saline controls were set. Each neutralization group and control group were inoculated with 5 duck embryos through the allantoic cavity, 0.2m1 / embryo, cultured at 37°C, observed for 7 days, and recorded the number of dead embryos in each group. The neutralization titer of the serum was defined as the highest serum dilution for 50% duck embryo protection.

[0099] The results of the neutralization test showed that the isolated strain could be completely neutralized by the positive serum of the isolated strain, but could not be neutralized an...

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Abstract

The invention provides a bivalent egg yolk antibody against DVH (duck virus hepatitis) as well as a preparation method and an application of bivalent egg yolk antibody. The bivalent egg yolk antibody contains a DHAV (duck hepatitis A virus)-1 type egg yolk antibody against DVH and a DHAV-3 type egg yolk antibody against DVH. The preparation method comprises steps as follows: (1) a DHAV-1 type strain against DVH and a DHAV-3 type strain against DVH are inoculated with an SPF chick embryo and a susceptible duck embryo respectively, an allantoic fluid is obtained, obtained virus fluids are mixed in proportion and inactivated with formalin, and a vaccine is prepared; (2) laying hens are immunized with the vaccine, sampling is performed after immunization for measuring whether the neutralizing titer of DHAV-1 type and DHAV-3 type antigens and antibodies in hyperimmune egg yolk of chickens is larger than or equal to 1:8192, and later, hyperimmune eggs of the chickens are collected; (3) eggshells of the hyperimmune eggs are disinfected, isovolumetric distilled water is added after the egg yolk is collected, and the mixture is stirred and mixed uniformly and then is subjected to pasteurization at the low temperature; purification with an acidified distilled water method and purification with a caprylic acid method are performed; microfiltration and ultrafiltration are performed. The provided bivalent egg yolk antibody is low in cost and high in titer, DVH caused by DHAV-1 and DHAV-3 can be effectively controlled, and remarkable social benefits can be obtained.

Description

[0001] This application is a divisional application of patent application 201210530208.5. The original invention title is "Duck Viral Hepatitis Bivalent Egg Yolk Antibody and Its Preparation Method and Application", and the filing date is December 11, 2012. technical field [0002] The invention relates to a hyperimmune egg yolk antibody, in particular to an egg yolk antibody for duck viral hepatitis. Background technique [0003] Duck Virus Hepatitis (DVH) is an acute, highly fatal and severe infectious disease of ducklings caused by Duck Hepatitis Virus (DHV) of the family Picornaviridae Enterovirus. The disease occurs all the year round and only infects ducklings within 3 weeks of age in a natural state, often causing significant economic losses and threatening the healthy development of the duck industry. The World Organization for Animal Health (OIE) classifies duck viral hepatitis as a category B animal disease. [0004] Duck hepatitis virus has historically been divi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/41C07K14/085C07K16/10C07K16/02A61K39/42A61K39/125A61P31/14
CPCA61K2039/505A61K2039/5252A61K2039/552C07K14/005C07K16/02C07K16/1009C07K2317/11C12N7/00C12N2770/32421C12N2770/32422C12N2770/32434
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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