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Updated: May 23, 2026
A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction
Published on: May 17, 2024
[Cloning and expression of gp37 gene of avian leukosis virus subgroup J]
Xiao-Wei Wang1, Qing Liu, Qing-Qing Xu
1College of Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China. wangxiaowei1209@126.com
Abstract:
The transmembrane protein (TM) encoded by gp37 gene plays a critical role when virus fusion with cell membrane occurs. Several highly conserved regions in TM are important targets for antivirus studies. Studies on structure and function of TM will provide basic information for anti-retrovirus, especially for avian leukosis virus. In the study, gp37 gene was amplified by PCR from the Chinese strain ALV-J-WS0701. The gp37 gene was cloned into pMD18-T vector, and was sequenced. Then, pFast-BacHTb-gp37 vector was constructed and expressed by baculovirus expression vector system. The expression product of gp37 gene was analyzed by indirect immunofluorescence assay and Western blot. The results showed that positive green fluorescence was present in sf9 cells infected with recombinant virus and a protein band with a molecular weight of 21kD was present in Western blot. It is concluded that gp37 gene was expressed in sf9 cells infected with recombinant virus successfully.
Insights
The gp37 gene, crucial for virus-cell fusion, was successfully expressed in sf9 cells using a baculovirus system. This achievement provides a foundation for developing antivirals targeting avian leukosis virus.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Context:
- The gp37 gene encodes a transmembrane protein (TM) vital for viral entry into host cells.
- Conserved regions within TM are key targets for antiviral drug development.
- Understanding TM's structure-function is essential for creating anti-retrovirals, particularly for avian leukosis virus (ALV).
Purpose:
- To amplify and clone the gp37 gene from the ALV-J-WS0701 strain.
- To express the gp37 gene in insect cells using a baculovirus expression system.
- To confirm the successful expression of the gp37 gene product.
Summary:
- The gp37 gene was amplified via PCR and cloned into a pMD18-T vector.
- A pFast-BacHTb-gp37 vector was constructed for expression in sf9 cells.
- Indirect immunofluorescence and Western blot confirmed successful expression, showing green fluorescence and a 21kD protein band.
Impact:
- Demonstrates successful expression of the avian leukosis virus gp37 gene in sf9 cells.
- Provides a basis for further structural and functional studies of the TM protein.
- Contributes to the development of novel antiviral strategies against avian leukosis virus.
