Method for preparing soluble human recombinant MICA protein

Abstract

The invention discloses a method for preparing a soluble human recombinant MICA protein, which comprises the following steps of: a step 1 of sampling 30ml of in-vitro peripheral venous blood donated by a healthy adult volunteer, diluting the in-vitro peripheral venous blood by physiological saline and separating out an offwhite lymphocyte layer by a lymphocytes separation medium; a step 2 of extracting an RNA (Ribose Nucleic Acid) of processed offwhite lymphocytes by a TRIZOL, carrying out sequence screening on the RNA, planting the RNA into a rhabdovirus genome, carrying out recombination, collection, dialysis and centrifugation to obtain an MICA protein and filtering the MICA protein by a CENTRICON ultrafilter; a step 3 of purifying a target protein; and a step 4 of identifying the MICA target protein. The soluble human recombinant MICA protein prepared by the preparation method is the MICA protein of which a molecular structure domain is closer to the natural state and has the advantages of convenience, rapidness, sensitivity, good repetitiveness and the like. Due to successful application of the soluble human recombinant MICA protein in an in-vitro immunological rejection model, practical support is provided and a solid foundation is laid for in-vivo experiments and clinical application of animals.

Description

technical field [0001] The invention relates to a preparation method of MICA protein, in particular to a preparation method of soluble human recombinant MICA protein. Background technique [0002] The immune rejection of grafts has been the biggest bottleneck that has plagued scholars at home and abroad since the emergence of organ transplantation. Although the research on the immune mechanism of transplantation is deepening and a large number of new immunosuppressive drugs are emerging, the probability of immune rejection of grafts after transplantation However, the use of immunosuppressants is expensive, and long-term use of immunosuppressants will greatly increase the chances of tumors and infections. Therefore, research on transplantation immunity is still one of the most critical topics. [0003] For a long time, the academic circles have focused on the classic major histocompatibility complex (MHC), that is, MHC-I and MHC-II, and established a classic theory of immune ...

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Problems solved by technology

[0005] Recently, American scholars have found MICA antibodies in the serum of some patients before and after kidney transplantation, and later found a higher titer of the antibody in the transplanted kidneys that had been rejected, suggesting that these patients have a higher titer in the kidneys. There is presensitization of allotype MICA antigen in the body before transplantation, and MICA antibody may play an important role in the rejection of kidney transplantation, but the mechanism of its participation in the immune response is still unclear

In addition, foreign studies on the MICA gene and its expression product MICA protein are still mainly focused on the field of kidney transplantation, while for other organ transplants, such as the impact of MICA protein on immune rejection after liver transplantation and its mechanism of action are still less

[0006] To sum up, there are still some gaps in the research on transplant immunity at home and abroad: ① Under the condition of blocking the classic MHC molecules of transplants, some transplants still have acute and chronic rejection reactions, indicating that there are other unknown factors causing or participate in the reaction, and the MICA gene may be one of the more important factors

②Although anti-MICA antibodies have been found in some organs before and after kidney transplantation and in the lavage fluid of organs with immune rejection, the mechanism of transplant immune rejection involving the MICA gene is still unclear, and most of the current research focuses on renal transplantation. Cellular and molecular levels of transplantation, lack of in vivo model studies in other organs such as liver transplantation and large animals

To fill the above gap, further research on the MICA gene is needed. However, the insoluble human recombinant MICA gene synthesized by the existing technology has fatal defects such as low sensitivity and single application method, which restricts the research work on the MICA gene.

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