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CN-122012399-A - HEK293 cell for stably expressing pigeon adenovirus type I Fiber2 protein, construction method and application thereof

CN122012399ACN 122012399 ACN122012399 ACN 122012399ACN-122012399-A

Abstract

The invention discloses HEK293 cells stably expressing pigeon adenovirus type I Fiber2 protein, and a construction method and application thereof. The cell QYH-PAF2-A1 capable of expressing the pigeon adenovirus type I Fiber2 protein is obtained by constructing a pigeon adenovirus type I Fiber2 protein recombinant expression vector and introducing into HEK293 adherent cells, and is domesticated into a suspension cell QYH-PAF2-A1. The cell has good suspension growth state and stable passage, and can stably express the Fiber2 protein after continuous culture for 10 generations, and then the Fiber2 protein expressed by the cell is prepared into subunit vaccine, and the protection rate reaches 80% through an immune toxicity attack test, so that the pigeon adenovirus I infection can be effectively prevented and controlled. The suspension cell constructed by the invention can be applied to research and development of pigeon adenovirus type I subunit vaccine, preparation of diagnostic reagent and basic research of virus invasion mechanism, and has wide application prospect.

Inventors

  • SUN XUE
  • WANG ZENGFU
  • LI YUE
  • WU ZONGXUE
  • MENG ZHAOYING
  • HUANG YUEQIANG

Assignees

  • 乾元浩生物股份有限公司

Dates

Publication Date
20260512
Application Date
20260121

Claims (9)

  1. 1. HEK293 cell QYH-PAF2-A1 for stably expressing pigeon adenovirus type I Fiber2 protein has a preservation number of CGMCC No.46766.
  2. 2. The method of constructing a cell according to claim 1, comprising the steps of: (1) Introducing a recombinant expression vector containing a pigeon adenovirus type I fiber2 gene into HEK293 adherent cells; (2) Screening to obtain positive clone cells capable of stably expressing Fiber2 protein; (3) And carrying out suspension domestication culture on the positive clone cells.
  3. 3. The method according to claim 2, wherein the recombinant expression vector in step (1) is constructed by cloning the pigeon adenovirus type I fiber2 gene into pcdna3.1 (+) vector.
  4. 4. The method of claim 2, wherein the recombinant expression vector is introduced into HEK293 adherent cells in step (1) using lipofection.
  5. 5. The method of claim 2, wherein the screening in step (2) comprises drug screening using G418 and monoclonalization using limiting dilution.
  6. 6. The method according to any one of claims 2 to 5, wherein the suspension acclimation culture in step (3) comprises a gradual decrease of serum concentration in the medium until serum-free culture.
  7. 7. Use of the cell of claim 1 for the preparation of pigeon adenovirus type I Fiber2 protein.
  8. 8. Use of the cell of claim 1 for the preparation of a subunit vaccine for preventing adenovirus type I infection in pigeons.
  9. 9. The use according to claim 8, wherein the pigeon adenovirus type I strain is QYH-HB-1 with a preservation number of CGMCC No.47084.

Description

HEK293 cell for stably expressing pigeon adenovirus type I Fiber2 protein, construction method and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to HEK293 cells stably expressing pigeon adenovirus type I Fiber2 protein, and a construction method and application thereof. Background The pigeon adenovirus I is an important pathogen causing clinical symptoms of vomiting, weakness, slow digestion, green stool or watery stool, weight loss and the like of pigeons, the disease spread speed is high, the incidence rate is 100 percent, the pigeons with weak resistance and mixed infection exist, the symptoms of physical performance decline, emaciation, listlessness, green stool or watery stool appear in the symptoms of frequent lasting months, and finally death occurs. The pigeon adenovirus type I Fiber2 protein is one of the main structural proteins of the virus, is positioned on the surface of virus particles, is a key antigen for inducing organisms to generate neutralizing antibodies, and has core value in vaccine preparation. At present, fiber2 proteins are mostly prepared by transient transfection or prokaryotic expression systems, but have the problems of low expression quantity, poor stability, incomplete protein modification and the like. The HEK293 cell strain has the advantages of strong suspension growth capability, high protein expression efficiency, easiness in large-scale culture and the like, and the construction of the HEK293 suspension cell strain for stably expressing the Fiber2 protein can realize the efficient and continuous expression of the target protein, thereby providing a key material for the research of pigeon adenovirus type I subunit vaccine. There is no report on such cell lines, so there is a need to develop a cell preparation technology with high efficiency and stability. Disclosure of Invention The invention aims to provide HEK293 cells stably expressing pigeon adenovirus type I Fiber2 protein, and a construction method and application thereof. In order to achieve the purpose of the invention, in a first aspect, the invention provides HEK293 cell QYH-PAF2-A1 which stably expresses pigeon adenovirus type I Fiber2 protein, the cell is preserved in China general microbiological culture Collection center, address Beijing Chaoyang area North Star West way No. 1, institute of microorganisms, post code 100101, preservation number CGMCC No.46766, and preservation date 2025, 12 months and 9 days. In a second aspect, the present invention provides a method of constructing the cell, comprising the steps of: (1) Introducing a recombinant expression vector containing a pigeon adenovirus type I fiber2 gene into HEK293 adherent cells; (2) Screening to obtain positive clone cells capable of stably expressing Fiber2 protein; (3) And carrying out suspension domestication culture on the positive clone cells. Further, the recombinant expression vector in the step (1) is constructed by cloning the pigeon adenovirus type I fiber2 gene into a pCDNA3.1 (+) vector. Further, in the step (1), the recombinant expression vector is introduced into HEK293 adherent cells by adopting a liposome transfection method. Further, the screening in step (2) includes drug screening using G418, and monoclonalization using limiting dilution. Further, the suspension acclimation culture in step (3) comprises gradually decreasing the serum concentration in the medium until serum-free culture. In a third aspect, the invention provides the use of said cells in the preparation of a pigeon adenovirus type I Fiber2 protein. In a fourth aspect, the invention provides the use of said cells in the preparation of a subunit vaccine for preventing adenovirus type I infection in pigeons. In the present invention, pigeon adenovirus (Aviadenovirus columbae) type I includes, but is not limited to, QYH-HB-1 strain which has been deposited at China general microbiological culture Collection center, address Beijing Kogyo North Star West Lu No. 1,3, institute of microorganisms, post code 100101, accession number CGMCC No.47084, date of deposit 2025, 11, 26 days. By means of the technical scheme, the invention has at least the following advantages and beneficial effects: The invention takes a mammalian cell HEK293 as a host cell, obtains a cell (QYH-PAF 2-A1) for stably expressing Fiber2 protein by a liposome transfection method, and acclimates the adherent cell into a suspension cell strain (QYH-PAF 2-A1). His tag is added at the C end of the Fiber2 gene, a nickel column is used for purifying protein, a pigeon adenovirus type I Fiber2 protein subunit vaccine is prepared, and an immune toxicity attack protection experiment is carried out. The cell strain can stably and continuously express the pigeon adenovirus I type protective antigen Fiber2 protein, the protein expression efficiency is obviously improved, and the protein produced by large-scale culture of the cell strain can pro