阅读说明：本技术 酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p抗原、抗体、制备方法和应用 (Saccharomyces cerevisiae calcium channel membrane protein Cch1p, Mid1p and Yvc1p antigens, antibodies, preparation method and application ) 是由 董晓宇 唐乾 王仁军 姜南 邱宇 于 2019-05-31 设计创作，主要内容包括：本发明涉及酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p抗原、抗体、制备方法和应用,属于基因工程技术领域。主要技术方案如下：酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p抗原的氨基酸序列分别为SED ID NO:1、SED ID NO:2、SED ID NO:3,基因序列分别为CCH1-300、MID1-190、YVC1-236。制备的三种抗体分别为Anti-Cch1p-300、Anti-Mid1p-190、Anti-Yvc1p-236。本发明为钙通道膜蛋白Western blot检测研究提供实验保障,也有利于解决目前缺少商品化抗钙通道膜蛋白抗体的问题。(The invention relates to saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p antigen, antibody, a preparation method and application, and belongs to the technical field of genetic engineering. The main technical scheme is as follows: the amino acid sequences of the saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p are SED ID NO 1, SED ID NO 2 and SED ID NO 3 respectively, and the gene sequences are CCH1-300, MID1-190 and YVC1-236 respectively. The prepared three antibodies are Anti-Cch1p-300, Anti-Mid1p-190 and Anti-Yvc1p-236 respectively. The invention provides experimental guarantee for Western blot detection and research of the calcium channel membrane protein, and is also beneficial to solving the problem that a commercial anti-calcium channel membrane protein antibody is lacked at present.)

1. The preparation method of the saccharomyces cerevisiae calcium channel membrane protein Cch1p, Mid1p and Yvc1p antibodies is characterized by comprising the following steps:

(1) determining amino acid sequences SED ID NO 1, SED ID NO 2 and SED ID NO 3 suitable for serving as antigen fragments; deducing gene sequences CCH1-300, MID1-190 and YVC1-236 according to the amino acid sequences;

(2) carrying out recombinant expression on proteins in Cch1p-300, Mid1p-190 and Yvc1p-236 regions of saccharomyces cerevisiae by using a prokaryotic expression system;

(3) by using Ni²⁺-obtaining recombinant proteins by NTA resin affinity chromatography;

(4) three recombinant purified proteins Cch1p-300, Mid1p-190 and Yvc1p-236 are respectively used for immunizing a mouse, an antibody is prepared by a cell fusion technology, and 3 strains of antibodies with high titer are obtained after screening and purification.

2. The saccharomyces cerevisiae calcium channel membrane protein Cch1p, Mid1p and Yvc1p antibodies prepared by the method of claim 1 are Anti-Cch1p-300, Anti-Mid1p-190 and Anti-Yvc1p-236, respectively.

3. The application of the saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p antibody in Western blot detection.

4. Saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p antigens, and is characterized in that the gene sequences are CCH1-300, MID1-190 and YVC1-236 respectively.

5. The Saccharomyces cerevisiae calcium channel membrane protein Cch1p, Mid1p, and Yvc1p antigens of claim 4, wherein the amino acid sequences are SED ID NO 1, SED ID NO 2, and SED ID NO 3, respectively.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p antigen, antibody, a preparation method and application.

Background

Saccharomyces cerevisiae is an important industrial microorganism for producing ethanol, and a metabolite thereof, namely bioethanol, is an important petroleum substitute; in addition, Saccharomyces cerevisiae is also a eukaryotic model bacterium. Therefore, the research on the calcium ion channel regulation mechanism is not only beneficial to improving the ethanol production capacity of the thalli, but also lays a foundation for the metabolism research of other eukaryotes.

The saccharomyces cerevisiae calcium channel membrane protein mainly comprises a voltage-gated calcium channel membrane protein (Cch1p), a stretch-sensitive calcium channel membrane protein (Mid1p) and a transient receptor potential calcium channel membrane protein (Yvc1p), wherein the former two regulate the intracellular calcium influx into cytoplasm, and the latter regulates the intracellular calcium efflux into cytoplasm in vacuole.

With the development of molecular biology and biochemistry, Western blotting (Western blot) is increasingly used for the study of ion channel regulation mechanisms. The Western blot technique is a protein detection technique for detecting a specific antigen by using a specific antibody, so as to obtain the information of the expression of the specific protein in the analyzed cells or tissues. However, in Western blot study of Saccharomyces cerevisiae calcium channel membrane protein, no commercial membrane protein antibody is reported. Therefore, the production of high quality antibodies is a crucial basis for carrying out this study.

Antibodies (abs) refer to glycoproteins produced by an organism after stimulation by an antigen, by B cells through proliferation and differentiation into plasma cells. Antibodies are classified into polyclonal antibodies and monoclonal antibodies according to the number of epitopes recognized by the antibodies. The monoclonal antibody has the characteristics of high specificity, strong affinity and the like, and is widely applied in the field of life science.

Disclosure of Invention

The invention provides the saccharomyces cerevisiae calcium channel membrane protein Cch1p, Mid1p and Yvc1p antigens, antibodies, a preparation method and application, provides experimental guarantee for Western blot detection research of the calcium channel membrane protein, and is also beneficial to solving the problem that commercial anti-calcium channel membrane protein antibodies are lacked at present.

The technical scheme of the invention is as follows: the preparation method of the saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p monoclonal antibodies comprises the following steps:

(1) determining amino acid sequences SED ID NO 1, SED ID NO 2 and SED ID NO 3 suitable for serving as antigen fragments; deducing gene sequences CCH1-300, MID1-190 and YVC1-236 according to the amino acid sequences;

(2) carrying out recombinant expression on proteins in Cch1p-300, Mid1p-190 and Yvc1p-236 regions of saccharomyces cerevisiae by using a prokaryotic expression system;

(3) by using Ni²⁺-obtaining recombinant proteins by NTA resin affinity chromatography;

(4) three recombinant purified proteins Cch1p-300, Mid1p-190 and Yvc1p-236 are respectively immunized for mice, monoclonal antibodies are prepared by a cell fusion technology, and 3 monoclonal antibodies with high titer are obtained after screening and purification.

The preparation method specifically comprises the following steps:

(1) respectively taking saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p as antigens to immunize a mouse;

(2) preparing immune spleen cells from the spleen of a mouse, mixing the immune spleen cells and SP2/0 cells in a centrifuge tube, fusing the cells by using polyethylene glycol to obtain hybridoma cells, and performing intraperitoneal injection on the hybridoma cells to produce ascites;

(3) and (4) passing the ascites through a purification column to obtain an eluent, and adding the eluent into the purification column for secondary purification to obtain the antibody.

Further, the method for immunizing the mouse in the step (1) specifically comprises the following steps:

the immune dose of the saccharomyces cerevisiae calcium channel membrane protein Cch1p antigen is 25 mug/body, and the immunization is performed once every two weeks for four times;

the primary dose of the antigen of the saccharomyces cerevisiae calcium channel membrane protein Mid1p is 100 mug/mouse, then the primary dose of each immunization is 50 mug/mouse, the immunization is carried out once every two weeks, and the mice are subjected to impact immunization once again 3 days before cell fusion for four times;

the vacuolar membrane calcium channel membrane protein Yvc1p antigen immunization priming dose is 100 mug/mouse, then each immunization dose is 50 mug/mouse, the immunization is carried out once every two weeks for four times, and the mice are subjected to impact immunization once again 3 days before cell fusion.

Further, the ratio of the immune spleen cells and SP2/0 cells in the step (2) is 5: 1.

Further, the step (3) is as follows: and (3) passing the ascites through a purification column to obtain an eluent, adding the eluent into the purification column, sealing the lower end of the purification column, and collecting the eluent by using a centrifugal tube filled with a neutralizing solution to obtain the antibody.

The invention simultaneously claims and protects the saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p monoclonal antibodies which are Anti-Cch1p-300, Anti-Mid1p-190 and Anti-Yvc1p-236 respectively.

The invention simultaneously requests to protect the application of the saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p monoclonal antibodies in Western blot detection.

The invention simultaneously requests to protect the antigens Cch1p, Mid1p and Yvc1p of the calcium channel membrane protein of the saccharomyces cerevisiae, and the gene sequences are CCH1-300, MID1-190 and YVC1-236 respectively.

Furthermore, the amino acid sequences of the saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p are SED ID NO 1, SED ID NO 2 and SED ID NO 3 respectively.

Furthermore, the saccharomyces cerevisiae calcium channel membrane proteins Cch1p, Mid1p and Yvc1p antigens are expressed in escherichia coli cells by gene segments and are obtained by purification.

Further, the saccharomyces cerevisiae calcium channel membrane protein Cch1p, Mid1p and Yvc1p antigens have the preparation method that: transforming the recombinant plasmid with the target gene into an escherichia coli expression strain, culturing to a logarithmic phase, inducing by 0.5mM isopropyl beta-D-thiogalactoside, and centrifuging to collect cell sediment;

resuspending the collected cell pellet with disruption buffer, ultrasonically disrupting the cell pellet to obtain supernatant, and subjecting the supernatant to Ni²⁺Purifying NTA resin, and concentrating with PEG 20000 to obtain antigen.

The invention has the following beneficial effects: according to the invention, through the preparation of three anti-saccharomyces cerevisiae calcium channel membrane protein monoclonal antibodies, an experimental guarantee is provided for the detection and research of the calcium channel membrane protein Western blot, the problem that a commercial anti-calcium channel membrane protein antibody is lacked at present is solved, and the verification experiment result proves that the obtained three monoclonal antibodies have the characteristics of high titer and strong specificity.

Drawings

FIG. 1 shows the target gene of Saccharomyces cerevisiae RNA reverse transcription PCR amplification;

wherein: 1. CCH1-300, 926bp, 2, MID1-190, 570bp, 3, YVC1-236, 708bp, M, RealBand DNA molecular weight standard Marker 100.

FIG. 2 shows the objective genes amplified by colony PCR;

wherein: 1-1 to 3, CCH1-300, 926bp, 2-1 to 3, MID1-190, 570bp, 3-1 to 3, YVC1-236, 708bp, M and RealBand DNA molecular weight standard Marker (100 to 10000 bp).

FIG. 3 is the induced expression of proteins Cch1p-300, Mid1p-190 and Yvc 1-236;

wherein: A. cch1p-300, molecular weight of about 60kDa, B, Mid1p-190, molecular weight of 25kDa, C, Yvc1p-236, molecular weight of 30 kDa. 1. Inducing total protein, supernatant at 2 and 20 ℃, intracellular protein at 3 and 20 ℃, supernatant at 4 and 37 ℃, intracellular protein at 5 and 37 ℃, and M and protein standard (14.4-116 kDa) before induction.

FIG. 4 is SDA-PAGE of purified recombinant proteins Cch1p-300, Mid1p-190 and Yvc 1-236;

wherein: 1. cch1p-300, molecular weight of about 60kDa, 2, Mid1p-190, molecular weight of 25kDa, 3, Yvc1p-236, molecular weight of 30kDa, M, protein standard (14.4-116 kDa).

FIG. 5 is a Western blot of recombinant purified proteins Cch1p-300, Midp1-190 and Yvc1 p-236;

wherein: 1. cch1p-300, molecular weight of about 60kDa, 2, Mid1p-190, molecular weight of 25kDa, 3, Yvc1p-236, molecular weight of 30kDa, M, protein standard (10-180 kDa).

FIG. 6 shows the mass spectrometric detection results of recombinant purified protein Cch1 p-300.

FIG. 7 shows WB analysis of purified membrane proteins expressed by recombinant clones and corresponding home-made monoclonal antibodies;

wherein: 1. Anti-Cch1p-300 with molecular weight of about 60kDa, 2, Anti-Mid1p-190, molecular weight of 25kDa, 3, Anti-Yvc1p-236, molecular weight of 30kDa, M, Marker (25-130 kDa).

FIG. 8 is a Western blot analysis of Saccharomyces cerevisiae cellular calcium channel membrane proteins;

wherein: 1. cch1p (234.6kDa), 2, Mid1p (61.5kDa), 3, Yvc1p (78kDa), M, Marker.

Detailed Description

The invention will be further illustrated with reference to specific examples: