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Cloning expression and polyclonal antibody preparation of black-headed gull IFN alpha protein

A black-billed gull and α-protein technology, applied in the field of bioengineering, can solve problems such as unreported research on wild migratory birds

Pending Publication Date: 2020-11-13
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IFNα products are widely used clinically, and there are many studies on mammals and poultry, but studies on wild migratory birds such as red-headed gull IFNα have not been reported

Method used

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  • Cloning expression and polyclonal antibody preparation of black-headed gull IFN alpha protein
  • Cloning expression and polyclonal antibody preparation of black-headed gull IFN alpha protein
  • Cloning expression and polyclonal antibody preparation of black-headed gull IFN alpha protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Analysis of Biological Information of Red-headed Gull IFNα

[0051] The protein analysis tool Protparam was used to analyze the basic physical and chemical properties of the protein; SignalP4.1 was used to predict the amino acid sequence signal peptide of the red-headed gull IFNα; the rare codons of the red-headed gull interferon-α gene were analyzed using the CBS website. The results are analyzed as follows:

[0052] (1) Using the protein analysis tool ProtParamα, the total number of amino acids in the protein is 192, the molecular weight of the protein is predicted to be about 21.93KD, and the theoretical value of the isoelectric point is 10.91. The atomic composition is C: 961; H: 1500; N: 312; O: 264; S: 9, the total number of atoms is 3046, and the molecular formula is C961H1500N312O264S9. The analysis of amino acid components shows that the amino acid with the highest content in this protein is Leu (14.1% ), Ala (9.9%), and His (9.9%). And the amino a...

Embodiment 2

[0056] Example 2: Cloning of partial fragments of IFNα of red-billed gull and construction of expression vector

[0057] 2.1 Extraction of total RNA from peripheral blood lymphocytes of red-headed gull

[0058] (1) Separation of blood lymphocytes

[0059] Lymphocytes were separated according to the instructions of the lymphocyte separation tube, and the cell solution was adjusted to 5x10 with incomplete RPMI1640 (containing 10% calf serum culture medium). 6 cells / ml, added to 6-well cell culture plate. 1600ul / well, 3 gradients for each sample (distributed into 6 wells, the surrounding wells cannot be added, otherwise it will be polluted), then add concanavalin A 80ul / well with a concentration of 5mg / ml, and place at 37°C, 5% CO2 incubator culture. After 24 hours of culture, the cells in the growth phase were inoculated in a 96-well cell culture plate (100ul / well cell suspension, that is, the cells need to be digested); the interferon inducer polyIc was added, and the final ...

Embodiment 3

[0113] Example 3: Construction and protein expression of the prokaryotic expression vector of a partial fragment of the IFNα gene of the red-billed gull

[0114] 3.1 Construction of recombinant prokaryotic expression vector pET32a-IFNα-1

[0115] (1) Extraction and recovery of PET 32a(+) and pMD 18T-IFNα-1 plasmids

[0116] ①Cultivate PET 32a(+) and pMD 18T-IFNα-1 strains.

[0117] ② The plasmid PET 32a(+) was reacted with the recombinant plasmid pMD 18T-IFNα-1 according to the enzyme digestion system in Table 5 below at 37°C for 3 hours.

[0118] Table 5 Reaction system for enzyme digestion

[0119]

[0120] ③ Carry out gel recovery of the digested vector pET32a(+) and the target fragment IFNα-1.

[0121] (2) Construction of recombinant expression strains

[0122] ① Ligate pET32a(+) and the target fragment IFNα-1 in the system shown in Table 6 at 16°C for 6 hours.

[0123] Table 6 connection system

[0124]

[0125] ② conversion

[0126] See 2.3(4) for the transf...

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Abstract

The invention relates to cloning expression and polyclonal antibody preparation of black-headed gull IFN alpha protein and belongs to the technical field of bioengineering. The invention provides cloning expression and polyclonal antibody preparation of the black-headed gull IFN alpha protein. Part of fragments of the black-headed gull alpha interferon gene are cloned, a prokaryotic expression recombinant vector is constructed, recombinant protein is obtained to prepare a polyclonal antibody, a theoretical basis is provided for later researches on the antiviral activity of the polyclonal antibody and the antiviral mechanism of the black-headed gull alpha interferon gene, a new path is developed for prevention and control of epidemic diseases of black-headed gulls, and veterinary public health is deeply affected. When rabbits are immunized, a Freund's complete adjuvant and an incomplete adjuvant are selectively applied to be matched with protein injection, and the polyclonal antibody can be efficiently and rapidly prepared. The agar diffusion test proves that the recombinant protein of partial segment of the black-headed gull IFN alpha gene has favorable immunogenicity, and the titer of the antibody can reach 1: 8. The method can be well used for subsequent researches on the antiviral mechanism of the black-headed gull IFN alpha protein.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to cloning and expression of red-billed gull IFNα protein and preparation of polyclonal antibodies. Background technique [0002] Migration is an instinctive activity of migratory birds. In order to escape threats from the environment and climate, migratory birds choose to migrate to different places to survive. During migration, it is easy to carry pathogens including parasites, bacteria and viruses. Birds, as carriers of pathogens, also pose potential threats to the survival of other vulnerable and endangered birds and even human health around their habitats. It has been reported recently that the outbreak of avian influenza is closely related to the migration of migratory birds. So far, avian influenza has been isolated from hundreds of wild birds. In addition to avian influenza, migratory birds will also carry other pathogens such as avian influenza and bronc...

Claims

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Application Information

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IPC IPC(8): C07K14/56C12N15/21C12N15/70C07K16/24C07K16/06
CPCC07K14/56C12N15/70C07K16/249C07K2317/10
Inventor 常华项勋代飞燕段纲杨林富段博芳曾邦全
Owner YUNNAN AGRICULTURAL UNIVERSITY
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