阅读说明：本技术 一种以Chitosan多糖为骨架制备MHC抗原肽多聚体检测试剂的新方法 (Novel method for preparing MHC antigen peptide multimer detection reagent by using Chitosan polysaccharide as skeleton ) 是由 洪建� 于 2018-09-06 设计创作，主要内容包括：抗原特异T细胞是免疫应答中的主要参与细胞,它参与免疫效应和调节免疫反应的进程。临床上在自身免疫疾病,抗肿瘤免疫反应,过敏变态反应等疾病过程中,抗原特异T细胞是重要的病理指标,可以与自身抗体同时检测,在某些自身抗体不明确的特殊疾病中更是最直接相关的免疫反应指标。本专利设计通过以多糖chitosan为骨架,标记上NTA-Ni镍离子,然后用基因重组方法合成带有Histidine的MHC class I重链,与β2m结合后,装上抗原表位肽(epitope peptide)形成复合体。将MHC/peptide复合体与骨架连接,由于一个NTA只结合2个位点,所以NTA-Ni上有2个空的Histidine结合位置,一个骨架分子上可以连接20-30个MHC/peptide复合体分子。定量快速检测抗原特异T细胞的数量,也可以用于体外分离抗原特异T细胞。具有检测灵敏度高,结合力强,背景低的特点。(Antigen-specific T cells are the major cells involved in the immune response, which are involved in the immune effect and in regulating the progress of the immune response. Clinically, in the disease processes of autoimmune diseases, anti-tumor immune reactions, allergic reactions and the like, the antigen specific T cells are important pathological indexes, can be simultaneously detected with autoantibodies, and are the most directly related immune reaction indexes in some specific diseases with unclear autoantibodies. According to the design, polysaccharide chitosan is used as a framework, NTA-Ni nickel ions are marked, then an MHC class I heavy chain with Histindine is synthesized by a gene recombination method, and after the MHC class I heavy chain is combined with beta 2m, epitope peptide (epitope peptide) is loaded to form a complex. The MHC/peptide complex is connected with the framework, and since one NTA only binds 2 sites, 2 empty Histidine binding sites are arranged on the NTA-Ni, and 20-30 MHC/peptide complex molecules can be connected with one framework molecule. The method can be used for quantitatively and rapidly detecting the number of the antigen specific T cells, and can also be used for separating the antigen specific T cells in vitro. The method has the characteristics of high detection sensitivity, strong binding force and low background.)

1. A method for chelating divalent nickel ion (Ni) by using Chitosan long sugar chain as skeleton and treating with nitrotricinic acid (NTA)²⁺) Forming MHC Class I/polypeptide epitope complex Chitosan-Ni-NTA for binding Histidine label.

2. A HisMHC/peptide multimer forms a polymerase detection reagent by the above Hi-tag and Chitosan-Ni-NTA-HisMHC/peptide.

3. A Chitin Binding Protein (CBP) of Chitosan-Ni-NTA-HisMHC/peptide polymer is detected by labeling various fluorescein (e.g., FITC, PE, APC, Percp, PE-Cy7, APC-Cy7, PE-Cy5.5, Percp-Cy5.5, etc.).

4. An assay method (including flow cytometric staining, immunofluorescent staining, etc.) for detecting antigen-specific T cells in human and animal blood using the above two reagents (Chitosan-Ni-NTA-HisMHC/peptide and fluorescein-labeled CBP).

Technical Field

The technical field of biological medicine.

Background

The antigen specific T cell is the main participating cell in immune response, and is involved in immune effect and the process of regulating immune response, and the detection of the antigen specific T cell is the most effective index method for responding to specific immune response. Clinically, in the disease processes of autoimmune diseases, anti-tumor immune reactions, allergic reactions and the like, the antigen specific T cells are important pathological indexes, can be simultaneously detected with autoantibodies, and are the most directly related immune reaction indexes in some specific diseases with unclear autoantibodies. Detection of antigen-specific T cells cannot be performed with pure antigen molecules, since the T Cell Receptor (TCR) on T cells recognizes only the MHC/peptide complex and cannot recognize a single pure antigen. Previous methods for detecting antigen-specific T cells include CTL killing assays, limiting dilution methods, intracellular cytokine staining, ELISPOT, all of which have limitations and are not directed to the observation of antigen-specific T cells, but are assessed by indirect effects of antigen-specific T cells. Detection of antigen-specific T cells using MHC/peptide multimers was first described in 1996 by the American Stanford university medical college, and currently methods for detecting antigen-specific T cells use MHC/peptide multimers, tetramer (tetramer), pentamer (pentamer) and long-chain dextramer multimers. Each tetramer and pentamer molecule contains four and five MHC/peptide complexes, respectively, and can bind to four and five T cell receptors, respectively, and sensitivity is limited for detecting antigen-specific T cells in low blood levels. The Dextramer contains 8-10 MHC/peptide complexes to improve sensitivity, but the stability of the reagent is also challenged, and the effective period of the reagent is not very long. Since the binding of a single TCR and MHC/peptide multimer is weaker, more TCRs bound can significantly improve the sensitivity of detection. Further, since antigen-specific T cells are generally present in small numbers in the blood, sufficient detection sensitivity is critical for successful detection.

Disclosure of Invention

According to the design, polysaccharide chitosan is used as a framework, NTA-Ni nickel ions are marked, then an MHC class I heavy chain with Histindine is synthesized by a gene recombination method, and after the MHC class I heavy chain is combined with beta 2m, epitope peptide (epiteptide) is loaded to form a complex. The MHC/peptide complex is connected with the framework, because one NTA is only combined with 2 sites, 2 empty Histidine combination positions are arranged on the NTA-Ni, and 20-30 MHC/peptide complex molecules can be connected with one framework molecule, thereby obviously improving the detection sensitivity. Chitosan is a stable sugar molecule, and the chelation of nickel ions is strong, so that the reagent prepared by the method is relatively stable. The patent is fundamentally different from the existing MHC/peptide multimer reagent, and the test reagent for the high-titer MHC/peptide multimer is prepared by taking chitosan as a framework and taking metal nickel ion chelation as a principle to load His MHC/peptide with a His label on the framework.

This patent is designed to use natural Chitin Binding Protein (CBP) as ligand, label various fluorescein (flurochrome) on the ligand protein, and use fluorescein labeled ligand to display antigen specific T cell bound by hisMHC/peptide multimer by flow cytometry. The binding of CBP and chitosan is very specific and the non-specific background staining is low compared to other binding based on biotin-streptavidin. In addition, the two have strong binding force and are not easy to dissociate.

The present patent discloses a novel method for preparing an antigen-specific T cell detection reagent by using hisMHC/peptide multimer and fluorescein-labeled CBP, which can prepare various MHC/peptide multimers, such as various MHC class I molecules (e.g., HLAA 0201, A1101, A2402, HLA B5801, B5201, etc.), and assemble various antigen polypeptides (e.g., CMV epitope polypeptide, EBV epitope polypeptide, HCV epitope polypeptide, HBV epitope polypeptide, etc.), based on the basic principle.

FIG. 1 is a schematic diagram of NTA-Ni and His-Tag binding. After NTA molecules are combined with nickel ions through chelation, one divalent nickel ion molecule can also be combined with two Histidine molecules.

FIG. 2 is a diagram of the molecular structure of Chitosan and N-succininyl Chitosan. The solubility of N-Succinyl Chitosan is higher than that of Chitosan, and the N-Succinyl Chitosan has wider application.

FIG. 3 is a schematic diagram of the hisMHC/peptide multimer structure. The gene recombinant protein is used for expressing an MHC Class I heavy chain containing His-Tag, and the His-Tag is combined at the C end. The β 2-microrogobulin is added by a protein renaturation process after purification of the recombinant protein.

FIG. 4 is a schematic representation of binding of hisMHC/peptide multimers to the N-Succinyl Chitosan polysaccharide backbone, pretreatment of the polysaccharide with NTA-Ni, and binding of fluorescein-labeled CBP to the Chitosan polysaccharide backbone. Whole molecules were used in flow cytometric staining to detect antigen-specific T cells in blood. Wherein the MHC Class I molecule and the polypeptide molecule can be replaced according to the antigen specific T cell to be detected and the individual MHC phenotype. CBP molecules are proteins and can label various fluorescein molecules, such as FITC, PE, PE-Cy5.5, Percp, APC, APC-Cy7, Percp-Cy5.5, PE-Cy7, and the like. According to the invention, Chitosan polysaccharide molecules are used as a framework for the first time, and through bivalent nickel ion mediation, Histidine modified MHC molecules are combined, and the CBP is labeled by fluorescein, so that the MHC polymer reagent for detecting antigen specific T cells is prepared.

Advantages or benefits of the invention

Compared with the prior art, the invention provides a novel MHC polymer reagent for detecting the number of antigen-specific T cells in blood. Based on the long-chain polysaccharide skeleton, each skeleton molecule can be combined with at least 20-30 hisMHC/peptide multimers which are 5-6 times more than the traditional tetramer, so the Chitosan-Ni-NTA-HisMHC/peptide multimer has high detection sensitivity and strong binding force, and because the CBP marked by fluorescein is used for coloring, the coloring background is low, and the specificity is high. In contrast, in the conventional biotin and streptavidin systems, there is always some background staining due to the presence of endogenous biotin. The Chitosan-Ni-NTA-HisMHC/peptide polymer prepared by the principle of the invention can be used in clinical detection to quantitatively and rapidly detect the number of antigen specific T cells and can also be used for in vitro separation of the antigen specific T cells.

Detailed description of the preferred embodiments and accompanying drawings

PBMC were obtained from 2 individuals (3X 10)⁶) After being suspended in 6-well cell culture plates with 10% FCS 1640, respectively, and stimulated with 30ug/ml CMV polypeptide pp65(NLVPMVATV) for 48 hours, stained with HLA A0201/pp 65(NLVPMVATV) and PE-labeled CBP, the content of stained CMV pp65 antigen-specific T cells was observed by flow cytometry analysis, and FIG. 5 is a flow chart showing the results of detection of specific antigen T cells using the present invention and PE-CBP.

The results show that when CD8+ cells were gated for PE fluorescence, PBMCs from two individuals contained 5.57% and 4.8% CMV pp65 antigen-specific T cells after antigen stimulation, respectively, indicating that HLA A0201/pp 65(NLVPMVATV) and PE-labeled CBP prepared using this inventive principle can detect antigen-specific T cells.

FIG. 6 is a flow chart for detecting antigen-specific T cells using the present invention. The detection process is as follows:

1) 1mL of venous blood is taken, 1mL of pbs is added, and after uniform mixing, the same volume of lymphocyte separation liquid is added.

2)400 g/min, centrifuging for 20 min, and taking a ring-shaped milky white lymphocyte layer.

3) Add 2mL pbs, wash cells, 300 g/min, centrifuge for 5 min. The supernatant was discarded.

4) The cells were resuspended in 50. mu.L pbs, an appropriate amount of Chitosan-Ni-NTA-HisMHC/peptide multimer was added, gently mixed, and incubated at room temperature for 30 min.

5) Add 2mL pbs qu buffer, place in centrifuge at 300 g/min, centrifuge for 5 min. The supernatant was discarded.

6) The cells were resuspended in 50. mu.L pbs, appropriate amounts of fluorescently labeled CBP and anti-CD 8 fluorescently labeled antibody were added, mixed gently and incubated for 10 min at room temperature in the dark.

7) Add 2mL pbs qu buffer, place in centrifuge at 300 g/min, centrifuge for 5 min. The supernatant was discarded. And repeating the steps once.

8) Cells were resuspended in 400. mu.L buffer and flow-loaded onto a machine.