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CN-121994763-A - Method for detecting binding capacity of poultry interferon and interferon receptor and application thereof

CN121994763ACN 121994763 ACN121994763 ACN 121994763ACN-121994763-A

Abstract

The invention belongs to the technical field of microbial genetic engineering, and discloses a method for detecting the binding capacity of poultry interferon and an interferon receptor in plants and application thereof, wherein the method comprises the following steps of S1, respectively constructing an optimized interferon gene and an interferon receptor gene to the N end and the C end of a plant expression vector to obtain a recombinant expression vector; S2, respectively converting the recombinant expression vectors into agrobacterium host bacteria to obtain genetically engineered bacteria, S3, injecting the genetically engineered bacteria into tobacco, culturing the tobacco injected with bacterial liquid to express the interferon and the interferon receptor in the tobacco, S4, capturing fluorescent signals by using a CCD imaging system, analyzing the fluorescent intensity of a specific area by using image analysis software, and detecting the interaction strength between the interferon protein to be detected and the interferon receptor protein by using the fluorescent intensity. The method is convenient to operate, visual in detection result, capable of greatly reducing workload and improving detection efficiency.

Inventors

  • XU XIAOXUE
  • ZHAO PENG
  • LIU XIAOJING
  • LIU ZHAOLU
  • YU LI

Assignees

  • 青岛英特凡恩生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260209

Claims (10)

  1. 1. A method for detecting the binding capacity of an avian interferon to an interferon receptor in a plant, said method comprising the steps of: S1, constructing an interferon recombinant expression vector, namely respectively constructing an interferon gene and an interferon receptor gene which are optimized according to codon preference of tobacco to the N end and the C end of a plant expression vector to obtain the interferon recombinant expression vector and the interferon receptor recombinant expression vector; S2, preparing genetically engineered bacteria, namely respectively converting the interferon recombinant expression vector and the interferon receptor recombinant expression vector into agrobacterium host bacteria to obtain the genetically engineered bacteria; s3, expressing the interaction protein in tobacco, namely injecting the genetically engineered bacterium into tobacco, and culturing the tobacco injected with bacterial liquid to express the interferon and the interferon receptor in the tobacco; S4, fluorescence imaging analysis, namely capturing a fluorescence signal by using a CCD imaging system, analyzing the fluorescence intensity of a specific area by using image analysis software, and detecting the interaction strength relationship between the interferon protein to be detected and the interferon receptor protein by using the fluorescence intensity.
  2. 2. The method for detecting binding capacity of avian interferon to interferon receptor in plants according to claim 1, wherein the optimization method of interferon gene sequence in step S1 is as follows: According to the codon preference of tobacco, carrying out codon optimization on an interferon protein coding gene to be detected, replacing a signal peptide coding gene sequence at the N end of the gene with a tobacco secretion signal peptide coding gene sequence, and removing a terminator at the tail end of the sequence to obtain the optimized interferon gene, wherein the amino acid sequence of the tobacco secretion signal peptide is shown as SEQ ID NO.23, and the nucleotide sequence is shown as SEQ ID NO. 24.
  3. 3. The method for detecting the binding capacity of avian interferon to an interferon receptor in plants according to claim 2, wherein in the step S1, the optimization method of the interferon receptor gene sequence is characterized in that the coding sequence corresponding to the 30 th to 243 th amino acids of the extracellular domain gene fragment of the interferon receptor protein coding gene is subjected to codon optimization according to the codon preference of tobacco, then a tobacco secretion signal peptide is added at the N end of the sequence, a terminator is removed at the end of the sequence, and the optimized interferon receptor gene is obtained, wherein the amino acid sequence of the tobacco secretion signal peptide is shown as SEQ ID No. 23.
  4. 4. The method for detecting the binding capacity of avian interferon and interferon receptor in plants according to claim 1, wherein the step S3 is that bacterial solutions of genetically engineered bacteria respectively transformed with an interferon recombinant expression vector and an interferon receptor recombinant expression vector are mixed, then the surfaces of tobacco leaves are subjected to perforation, the mixed bacterial solutions are injected on the surfaces of the leaves, and then the treated tobacco is subjected to normal temperature culture for 24-48 hours under dark conditions, and then illumination is recovered for 12-24 hours.
  5. 5. The method for detecting the binding capacity of avian interferon to interferon receptor in plants according to claim 1, wherein the step S4 comprises the steps of spraying luciferase on the surfaces of tobacco leaves, performing light-shielding treatment, capturing LUC images by using a precooled CCD imaging device, and obtaining the LUC images for 10S-1 min.
  6. 6. The method for detecting binding capacity of avian interferon to interferon receptor in plants according to claim 5, wherein the step S4 further comprises the step of transferring all fluorescence data to ImageJ software, quantitatively analyzing the fluorescence intensity of specific areas of the leaf, and reflecting the interaction strength relationship between interferon and interferon receptor according to the fluorescence intensity data.
  7. 7. The method for detecting the binding capacity of avian interferon to an interferon receptor in plants according to claim 3, wherein the interferon is chicken interferon alpha, the interferon receptor is chicken interferon alpha receptor, the amino acid sequence of the optimized chicken interferon alpha receptor is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO.2, or the amino acid sequence of the optimized chicken interferon alpha is shown as SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 or SEQ ID NO.3, preferably the nucleotide sequence of the optimized chicken interferon alpha is shown as SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10 or SEQ ID NO. 4.
  8. 8. A chicken alpha interferon receptor recombinant vector for detecting the binding capacity of avian interferon and an interferon receptor is characterized by being prepared by converting a chicken alpha interferon receptor gene sequence shown as SEQ ID NO.2 into an expression vector, wherein the expression vector is pCambia1300.
  9. 9. A chicken interferon alpha expression strain for detecting the binding capacity of avian interferon to an interferon receptor, which is transformed with the recombinant vector of claim 8, wherein the strain is agrobacterium.
  10. 10. The method for detecting binding capacity of avian interferon to interferon receptor in plants according to claim 1 to 7, the use of chicken interferon alpha receptor recombinant vector according to claim 8 or chicken interferon alpha expression strain according to claim 9 in high activity avian interferon screening.

Description

Method for detecting binding capacity of poultry interferon and interferon receptor and application thereof Technical Field The invention relates to the technical field of microbial genetic engineering, in particular to a method for detecting the binding capacity of poultry interferon and an interferon receptor and application thereof. Background The first large country of global poultry breeding is over 120 hundred million chickens, accounting for over 35% of the world, however, the virus diseases such as avian influenza, newcastle disease and the like cause direct economic loss per year. Chicken interferon is widely used for preventing and treating viral diseases of poultry at present. After general prohibition of growth-promoting antibiotics in the agricultural rural department 2020, interferon was incorporated as an alternative into the "veterinary antibacterial use reduction action regimen". Therefore, the research of the interferon is not only the strategic requirement of epidemic prevention and control, but also the industrial upgrading and international competition core gripper. Among them, chicken interferon alpha (chifnα) is an important antiviral cytokine in the immune system of birds, and through binding with the receptor (heterodimer formed by IFNAR1 and IFNAR 2) on the cell membrane, activates JAK-STAT signal pathway, induces the expression of a series of antiviral proteins (such as 2',5' -oligoadenylate synthetase), thereby inhibiting the proliferation of viruses such as avian influenza, newcastle disease and infectious bursal disease, and exerting antiviral effect. Researches show that the chicken alpha interferon can reduce the use amount of antibiotics by more than 50 percent, and simultaneously improve the culture benefit. The chicken alpha interferon is a green additive which is mainly promoted in Henan province, shandong province and the like, and enjoys research and development subsidies and tax benefits. The binding affinity of the wild-type ChIFNalpha and the receptor is limited, so that the antiviral efficiency of the wild-type ChIFNalpha is difficult to reach an ideal level, and the application of the wild-type ChIFNalpha in cultivation production is limited. The difficulty of developing and screening the interferon receptor with high binding affinity and high antiviral activity is high, and the conventional interferon antiviral activity detection method has the advantages of complex steps, large workload, long period and high cost, and also causes low screening efficiency of high-quality interferon. Thus, the existing methods for detecting the antiviral activity of avian interferon and screening methods for interferon have yet to be further improved. Disclosure of Invention Aiming at the problems, the invention provides a method for detecting the binding capacity of poultry interferon and an interferon receptor in plants and application thereof, which can greatly reduce the workload, improve the detection efficiency and shorten the period. In order to solve the problems, the application provides the following technical scheme: In a first aspect, the present application provides a method of detecting the binding capacity of an avian interferon to an interferon receptor in a plant, said method comprising the steps of: S1, constructing an interferon recombinant expression vector, namely respectively constructing an interferon gene and an interferon receptor gene which are optimized according to codon preference of tobacco to the N end and the C end of a plant expression vector to obtain the interferon recombinant expression vector and the interferon receptor recombinant expression vector. The step can adopt the designed and synthesized interferon genes and interferon receptor genes in advance to construct the recombinant expression vector of the interferon. The optimized design of the gene can also be carried out by the heavy head. S2, preparing genetic engineering bacteria, namely respectively converting the interferon recombinant expression vector and the interferon receptor recombinant expression vector into agrobacterium host bacteria to obtain the genetic engineering bacteria. S3, expressing the interaction protein in tobacco, namely injecting the genetically engineered bacterium into tobacco, and culturing the tobacco injected with the bacterial liquid to express the interferon and the interferon receptor in the tobacco. S4, fluorescence imaging analysis, namely capturing a fluorescence signal by using a CCD imaging system, analyzing the fluorescence intensity of a specific area by using image analysis software, and detecting the interaction strength relationship between the interferon protein to be detected and the interferon receptor protein by using the fluorescence intensity. The method for detecting the binding capacity of the poultry interferon and the interferon receptor in the plant is based on the screening and detection of the binding capacity between proteins by a lu