Method for preparing TW1 type avian infectious bronchitis virus positive serum from SPF chickens

A chicken infectious and positive serum technology, applied in the direction of chemical instruments and methods, antiviral immunoglobulin, immunoglobulin from serum, etc., can solve complex and variable biological characteristics, pathogenicity and tissue tropism, cross Protection and serum neutralization efficiency are poor, and disease diagnosis and prevention work are difficult, so as to achieve the effect of improving the level of prevention and control, controllable quality, and stable source

Active Publication Date: 2019-11-19
ZHONGCHONG XINNUO BIOTECH TAIZHOU CO LTD
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  • Application Information

AI Technical Summary

Problems solved by technology

The genomic nucleic acid of chicken infectious bronchitis virus (abbreviated as IBV) is prone to mutation and recombination during the replication process, and the biological characteristics, pathogenicity and tissue tropism of different strains are complex and changeable. Differences can divide IBV into numerous serotypes, and the cross-protection and serum neutralization efficiency between different serotypes of IBV are relatively poor, which brings great difficulties to disease diagnosis and prevention.

Method used

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  • Method for preparing TW1 type avian infectious bronchitis virus positive serum from SPF chickens
  • Method for preparing TW1 type avian infectious bronchitis virus positive serum from SPF chickens
  • Method for preparing TW1 type avian infectious bronchitis virus positive serum from SPF chickens

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Embodiment 1

[0039] Embodiment 1——Virus content and purity detection of virus species for immunization

[0040] Carry out virus content determination to virus species according to the following method, AH1103 chicken embryo allantoic fluid is made 10 times of serial dilutions with sterile saline, take 10 -5 、10 –6 、10 –7 、10 –8 For 4 dilutions, 5 10-day-old SPF chicken embryos were inoculated into each allantoic cavity, 0.1ml per embryo, and incubated at 36-37°C. Chicken embryos that died before 24 hours were discarded, and chicken embryos that died within 24 to 144 hours were taken out at any time, until 144 hours, all live embryos were taken out. The dead chicken embryos and surviving chicken embryos in 24-144 hours after inoculation have specific lesions such as dehydration, curling up, and small development (the weight of the inoculated fetus is more than 2g lower than that of the lightest fetus in the control) and other specific lesions are judged as infection, and the EID is calc...

Embodiment 2

[0045] Embodiment 2——Genotyping of chicken infectious bronchitis virus IBV CK / CH / XNGD / 140 strains

[0046] According to the sequence of chicken infectious bronchitis virus (IBV) S1 gene included in GenBank, primers (sequence 1 and sequence 2) were designed for the conserved region after alignment, and were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The primer sequences are as follows:

[0047] Sequence 1 upstream primer S1–F: 5′–atgttgggga agtcactgtt–3′20

[0048] Sequence 2 downstream primer S1–R: 5′–acgtctaaaa cgacggctt–3′19.

[0049] IBV CK / CH / XNGD / 140 strains were inoculated into 10-day-old SPF chicken embryos, and the allantoic fluid of 36-42h chicken embryos was collected. The S1 gene of CK / CH / XNGD / 140 strain was amplified by RT-PCR, and the amplified product was cloned, sequenced, and compared with the gene sequence. Viral RNA was extracted from allantoic fluid according to the instructions of the RNA extraction kit of Tiangen Biochemical Technology (Bei...

Embodiment 3

[0057] Embodiment 3——Preparation of chicken infectious bronchitis virus IBV CK / CH / XNGD / 140 strain inactivated vaccine

[0058] Inoculate 10-day-old SPF chicken embryos with CK / CH / XNGD / 140 virus seeds, and collect the allantoic fluid of 36-42h chicken embryos. The harvested chicken embryo virus liquid was centrifuged at 8000 rpm for 10 min, and the supernatant was harvested. The centrifuged virus fluid was dialyzed and concentrated to 1 / 5 of the original volume, and inactivated with formaldehyde at a final concentration of 0.15% at 37° C. for 20 h. Preparation of oil phase: heat white oil (94 parts) to 80°C, then add Siben-80 (6 parts), mix evenly, heat to 121°C for 30 minutes, cool for later use. Preparation of aqueous phase: add sterilized Tween-80 (4 parts) to inactivated antigen solution (96 parts), and mix well. Emulsification: Measure the water phase which is 1 / 3 of the volume of the oil phase, add it to the oil phase, and mix at 1500r / min for 2min. Adjust the speed to...

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Abstract

The invention discloses a method for preparing TW1 type avian infectious bronchitis virus positive serum from SPF chickens. An IBV virus seed of a specific genotype (TW1 type) is used. The method comprises the steps: the IBV CK/CH/XNGD/140 strain virus seed and an oil emulsion inactivated vaccine thereof are used for conducting basic immunization and enhanced immunization on SPF chickens which are50 days old, blood sampling is conducted, and serum is separated; and the TW1 type IBV positive serum with good specificity and high titer is obtained by carrying out sterile and exogenous virus detection, neutralization titer determination and specificity detection on the serum. The preparation method of the TW1 type IBV positive serum fills the blank that the TW1 type IBV virus seed has no standard positive serum. The SPF chickens are selected as test animals for preparing the serum, the immune procedure in the serum preparation process is optimized, and the serum antibody neutralization titer is improved. The production cost is reduced, and batch preparation of the positive serum is facilitated.

Description

technical field [0001] The invention relates to a method for preparing chicken infectious bronchitis positive serum by using SPF chickens, and belongs to the technical field of veterinary biological products. Background technique [0002] Chicken infectious bronchitis (Avian infection bronchitis) is an acute, highly contagious infectious disease caused by chicken infectious bronchitis virus (Avian infection bronchitis virus), which mainly affects the respiratory system, genitourinary system and digestive system of chickens Wait. The genomic nucleic acid of chicken infectious bronchitis virus (abbreviated as IBV) is prone to mutation and recombination during the replication process, and the biological characteristics, pathogenicity and tissue tropism of different strains are complex and changeable. Differences can divide IBV into numerous serotypes, and the cross-protection and serum neutralization efficiency between different serotypes of IBV are relatively poor, which brin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C07K16/06
CPCC07K16/10C07K16/06C07K2317/76C07K2317/33
Inventor 何海蓉陈启稳刁小龙印广浩张家铭陈坚颜文光
Owner ZHONGCHONG XINNUO BIOTECH TAIZHOU CO LTD
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