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Research on using insect cell expression system to express immune-related molecules

2021-04-29
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Both CD4 molecule and IL-12 are hot molecules in tumor immunotherapy research. Among them, CD4 molecule has important immunological functions, it can participate in the body’s immune response, and the cytokines of the IL-12 family of interleukins play a role in immune regulation. The immune response can be formed by directly inducing the development of T cell subpopulations and changing the functions and fate of many cell populations that determine the prognosis of the disease. Using protein expression systems such as insect cell expression systems to express biologically active CD4 and IL-12 molecules can provide a wealth of information for the treatment of immune diseases.

The CD4 molecule is a 55KD single-chain transmembrane glycoprotein, which plays an important role in the activation and signal transduction of T cells. Many diseases such as autoimmune diseases, transplant rejection and human immunodeficiency virus are related to CD4 molecules. Close connection. CD4 molecule has important immunological functions. It can participate in the body’s immune response and plays an important role in the body’s normal immune function. CD4 molecule has also become an important target for the treatment of many diseases. CD4 molecules can interact with MHC and TCR to form a CD4-MHC-TCR complex, mediate specific antigen presentation and activate CD4+ T cells, triggering a body-specific immune response. At the same time, CD4 is the main receptor on HIV-infected host cells, and CD4 fusion protein drugs and CD4 antibodies have shown good therapeutic effects in clinical studies of AIDS. CD4 protein expression through protein expression system such as insect cell expression system can provide a theoretical basis for studying its protein activity.

Some researchers have achieved high-efficiency expression of the extracellular domain of human CD4 protein in the baculovirus-insect cell expression system through optimization of conditions, and analyzed the antigenicity and immunogenicity of the purified extracellular domain of human CD4 protein. The researchers constructed a recombinant baculovirus (pAc-CD4) by selecting extracellular fragments of human CD4 molecules, and then infecting insect cells for protein expression. Purified by nickel ion affinity chromatography. Using SDS-PAGE, Western blot, and size exclusion Chromatography, ELISA, SPR methods, etc., analyze and purify the physical and chemical properties and antigenicity of the target protein. The BALB/c mice were immunized with Freund’s adjuvant and the target protein to monitor the antibody production of the mouse immune serum [1]. This research uses the baculovirus-insect cell system for high-efficiency expression to obtain human CD4 extracellular domain protein with good antigenicity and immunogenicity, which lays the foundation for the research of HIV receptor and infection.

Insect cell expression system is a widely used eukaryotic expression system, which can express a variety of foreign genes including fungi, plants, bacteria and viruses in insect cells. Baculovirus insect expression vectors have large capacity and can be used in a virus vector Simultaneously express two or more foreign genes on the computer. Medicilon researchers have established a mature baculovirus-insect cell expression service platform, providing services including the preparation of recombinant baculovirus, the expression and purification of recombinant proteins and their complexes.

Interleukin 12 (IL-12) is a heterodimeric cytokine mainly produced by antigen presenting cells (monocytes, macrophages, dendritic cells and B cells). Bacteria, bacterial secretions and intracellular parasites can stimulate the production of IL-12. Granulocyte-macrophage colony stimulating factor (GM-CSF) and γ-interferon (IFN-γ) can enhance the production of IL-12 . The main biological function of IL-12 is to promote their reproduction, produce cytokines and increase cytotoxicity by acting on T cells and natural killer (NK) cells. It acts as an inflammatory cytokine and an immunomodulator. Resistance and acquired immunity build a bridge.

Some researchers have established a baculovirus/insect cell expression system that expresses biologically active human IL-12. The researchers construct p40 and p35 cDNA together into the baculovirus transfer vector pAcUW42, and then combine it with the baculovirus AcUW1.lacZ DNA Co-transfect insect cells Sf9 to cause homologous recombination between the two; then use the virus plaque experiment to screen the recombinant baculovirus, then screen and identify the recombinant virus expressing IL-12 by ELISA, and perform Western Blotting and in vitro biological activity The detection [2]. As a result, this study established a baculovirus/insect expression system capable of expressing human recombinant IL-12 with in vitro biological activity.

Some molecules related to immune function play an important role in maintaining the body’s immune tolerance and immune balance. The imbalance of its immune suppression function is closely related to diseases or pathological conditions such as autoimmune diseases, tumors, inflammatory immune responses and transplant rejection. The use of insect cell expression systems to obtain biologically active proteins can lay the foundation for subsequent research on disease treatment.

[1] Expression, purification and characterization of human CD4 extracellular domain protein in baculovirus-insect cell system [J].

[2] Expression of human IL-12 in insect cells and detection of its in vitro biological activity [J].

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