Blood type reverse typing reagent, preparation method thereof and blood type reverse typing detection kit

Abstract

The invention discloses a blood type reverse typing reagent, a preparation method thereof and a blood type reverse typing detection kit. The method comprises the following steps: S1, constructing functional magnetic nanoparticles for expressing recombinant protein G; s2, preparing erythrocyte membrane antigen magnetic nanoparticles: adding an anti-H blood group monoclonal antibody into red blood cells, washing, splitting the red blood cells, adding the functionalized magnetic nanoparticles, washing, magnetically attracting, and freeze-drying a magnetic product to obtain the blood group reverse typing reagent. By utilizing the advantages of excellent magnetic properties and biocompatibility of magnetosomes and a surface display technology, protein G is expressed on the surfaces of the magnetosomes through genetic engineering and is specifically combined with an anti-H blood group antigen monoclonal antibody, and erythrocyte membrane antigens are coupled to the surfaces of the magnetosomes to form erythrocyte membrane antigen magnetic nanoparticles; and the problems of easy hemolysis of red blood cells and disappearance of red cell membrane antigens after long-time storage can be avoided. The magnetosome not only serves as a carrier of the antigen, but also serves as an indicator of the agglutination result, and result interpretation is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and in particular to a blood type reverse typing reagent, a preparation method thereof and a blood type reverse typing detection kit. Background Art

[0002] Blood transfusion is an important means of modern medicine. Blood type compatibility testing of recipients and donors (including positive and negative blood typing testing, antibody testing and cross matching) is one of the most important conditions for ensuring safe blood transfusion. Blood type incompatibility can cause hemolytic transfusion reactions, transplant organ rejection reactions and neonatal hemolytic disease. Among them, the key to blood type reverse typing testing lies in red blood cell blood type antigen reagents. Red blood cell blood type antigens are structural antigens. Some protrude on the cell surface, such as ABH antigens, and some are embedded in the cell membrane, such as Rh antigens. After hemolysis, these antigen structures are easily destroyed and lose their activity. At present, the long-term effective preservation of red blood cell blood type antigens has always been a thorny problem. The ABO reverse typing reagents used in clinical applications are all fresh red blood cells. Fresh red blood cells are easy to hemolyze and difficult to store. The validity period of cellular antigens is too short, and the quality of the reagents is difficult to guarantee. The stability period of reverse typing red blood cells prepared by clinical laboratories is mostly between 3 and 60 days, and the commercial reagent red blood cells on the market have a validity period of only 90 days. After the time of factory transportation and reagent supplier preparing goods, there is not much time left for the validity period to reach the user. The freshness of red blood cells within the validity period directly affects the antigen strength of red blood cells. The structure and stability of red blood cell membranes and membrane proteins will change with the increase of storage time. Hemolysis, weak agglutination, and false negative results are often seen in clinical applications, which seriously interfere with the results of blood typing tests. The unstable quality of red blood cell antigen reagents makes it difficult to ensure the accuracy of test results, which is also one of the reasons for the discrepancy between positive and negative typing in current clinical blood typing.

[0003] Hydroformylation of red blood cells can provide a certain degree of protection for the red blood cell membrane structure, but it will affect the antigen activity on the surface. Some blood type reference laboratories use cryopreservation or freeze-drying to preserve red blood cells, but cryopreservation is time-consuming and energy-consuming, and inconvenient to transport; and freeze-dried red blood cells will cause cell damage when they are rehydrated, resulting in high cell loss and low recovery rate. In addition, washing to remove the protective agent is cumbersome and difficult to apply on a large scale.

[0004] In view of the problem of natural red blood cell antigens being difficult to preserve, in recent years, as people have a very clear understanding of the composition and structure of most blood type antigen encoding genes, antigen molecules and antigen carriers, the study of blood type antigens that do not rely on active human red blood cells has become one of the key research directions in the field of blood immunology. Blood type antigens such as Rhesus, Duffy and Kidd have been successfully expressed in eukaryotic cell lines in a membrane-bound form and have been used for the detection of corresponding antibodies. However, compared with protein antigens, the in vitro recombination of carbohydrate antigens such as blood type A and B is much more difficult. Through the random peptide library displayed on the phage surface, A and B blood type monoclonal antibodies were used as solid phase screening targets, and some mimetic epitopes of A and B blood type antigens have been screened. Agglutination inhibition tests show that A and B blood type carbohydrate antigens can use peptide molecules to simulate antigen epitopes to specifically bind to anti-A and anti-B. However, the antigenicity of artificial blood type antigens prepared by methods such as recombinant antigens and mimetic peptides is different from that of natural antigens, and erroneous results are prone to occur during clinical testing. The method of use is also quite different from the conventional clinical method, making it difficult to promote. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a blood type reverse typing reagent, a preparation method thereof and a blood type reverse typing detection kit in view of the deficiencies in the above-mentioned prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: in the first aspect of the present invention, a method for preparing a blood type reverse typing reagent is provided, comprising the following steps:

[0007] S1. Construction of functionalized magnetic nanoparticles expressing recombinant protein G;

[0008] S2. Preparation of red blood cell membrane antigen magnetic nanoparticles:

[0009] Anti-H blood type monoclonal antibody is added to the red blood cells, the red blood cells are lysed after washing, and then the functionalized magnetic nanoparticles prepared in step S1 are added, washed, magnetically adsorbed, and the magnetic product is freeze-dried to obtain red blood cell membrane antigen magnetic nanoparticles, i.e. the blood type reverse typing reagent.

[0010] Preferably, step S1 specifically includes:

[0011] S1-1, construction of a mutant strain of magnetotactic bacteria MSR-1 lacking the bacterial magnetic particle membrane proteins mamC / mamF;

[0012] S1-2, construct functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G;

[0013] S1-3. The functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G is transferred into the magnetotactic bacteria MSR-1 mutant strain lacking the bacterial magnetic particle membrane protein mamC / mamF gene obtained in step S1 through conjugation to construct a magnetotactic bacteria MSR-1 recombinant strain, culture it, and prepare functional bacterial magnetic particles.

[0014] Preferably, step S1-1 specifically includes:

[0015] S1-1-1, amplify two homologous fragments of 700-1000 bp in length on both sides of the bacterial magnetic particle membrane protein gene; both homologous fragments have two restriction sites;

[0016] S1-1-2, the left and right homologous fragments were double-digested with the corresponding restriction endonucleases, and the suicide plasmid pK18mobsacB was double-digested at the same time;

[0017] S1-1-3, connect the left and right homologous fragments after double enzyme digestion and the suicide plasmid after double enzyme digestion, transfer the connection products into E. coli competent cells, and select the positive clones with correct connection;

[0018] S1-1-4, using the positive clone bacteria as donors to carry out parental conjugation with the magnetotactic bacteria MSR-1 recipient bacteria, and introducing the pK18mobsacB recombinant plasmid, screening the magnetotactic bacteria MSR-1 homologous integration mutants through kanamycin resistance, and then screening the magnetotactic bacteria MSR-1 double exchange mutants through sucrose gradient, and finally selecting the kanamycin resistance-sensitive double exchange strains for verification, and constructing the magnetotactic bacteria MSR-1 mutant strains with the bacterial magnetic particle membrane protein mamC / mamF gene deleted;

[0019] Preferably, step S1-2 specifically includes:

[0020] S1-2-1, amplify the DNA fragment of the transmembrane domain of the bacterial magnetic particle membrane protein mamC / mamF gene, the two ends of the DNA fragment respectively have double enzyme cutting sites; perform double enzyme cutting on the amplified product DNA fragment and the pBBR plasmid, recover the double enzyme cutting DNA fragment and the double enzyme cutting pBBR plasmid; connect the double enzyme cutting DNA fragment with the double enzyme cutting pBBR plasmid, transform the connection product into Escherichia coli competent cells, screen the positive clones with correct connection, and obtain the recombinant plasmid pBBR-bacterial magnetic particle membrane protein mamC / mamF gene;

[0021] S1-2-2, clone the coding sequence of recombinant protein G into the pUC57 vector, and add double restriction sites at both ends;

[0022] S1-2-3, amplifying and double-enzyme digesting the recombinant protein G plasmid obtained by gene synthesis in step S1-2-2;

[0023] S1-2-4, double-enzyme digestion and purification of the recombinant plasmid pBBR-bacterial magnetic particle membrane protein mamC / mamF gene obtained in step S1-2-1, and ligation with the gene fragment of the double-enzyme digested polypeptide chain-specific recombinant protein plasmid obtained in step S1-2-3, the ligation product is transformed into Escherichia coli competent cells, and the positive clones with correct ligation are screened and verified, and finally the functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR-bacterial magnetic particle membrane protein gene-recombinant protein G is obtained;

[0024] Preferably, step S1-3 specifically includes:

[0025] S1-3-1, using the functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G as a donor, it was introduced into the magnetotactic bacteria MSR-1 mutant strain lacking the bacterial magnetic particle membrane protein mamC / mamF gene through a parental conjugation experiment to construct a magnetotactic bacteria MSR-1 recombinant strain;

[0026] S1-3-2. The recombinant strain of magnetotactic bacteria MSR-1 is transferred and fermented, and the functional bacterial magnetic particles prepared by bacterial expression are determined by a magnetic device. The bacteria are collected by centrifugation and extracted and purified to obtain functional bacterial magnetic particles.

[0027] Preferably, step S2 specifically includes:

[0028] S2-1. Collect fresh red blood cells, wash them three times with 0.9% NaCl solution, and discard the supernatant;

[0029] S2-2, add IgG anti-H blood group monoclonal antibody, incubate at 37°C for 30 minutes, wash three times with 0.9% NaCl solution, and discard the supernatant;

[0030] S2-3, add a 0.6% NaCl solution of equal volume to the packed red blood cells, mix well, keep at room temperature for 5 minutes, centrifuge at 2000 rpm for 5 minutes and collect the precipitate;

[0031] S2-4, adding a 0.2% NaCl solution of the same volume as the packed red blood cells to the precipitate obtained in step S2-3, mixing and keeping at room temperature for 5 minutes, centrifuging at 3000 rpm for 10 minutes and collecting the precipitate;

[0032] S2-5, adding distilled water of the same volume as the packed red blood cells to the precipitate obtained in step S2-4, mixing and keeping at room temperature for 5 minutes;

[0033] S2-6, adding the functionalized magnetic nanoparticles prepared in step S1-1, and incubating at 37° C. for 30 minutes;

[0034] S2-7, magnetic adsorption, the magnetic product was washed three times with PBS;

[0035] S2-8, adding lyophilization protection solution to the product of step S2-7, resuspending, packaging and lyophilizing to obtain red blood cell membrane antigen magnetic nanoparticles, i.e., the blood group reverse typing reagent;

[0036] Among them, red blood cells include type A red blood cells, type B red blood cells, and type O red blood cells, and the corresponding prepared red blood cell membrane antigen magnetic nanoparticles are type A red blood cell membrane antigen magnetic nanoparticles, type B red blood cell membrane antigen magnetic nanoparticles, and type O red blood cell membrane antigen magnetic nanoparticles.

[0037] Preferably, the freeze-dried protective solution comprises the following components by mass concentration: 5% sucrose, 1% PEG4000, 1% bovine serum albumin, 3.58% disodium hydrogen phosphate dodecahydrate, 0.201% potassium chloride, 8.19% sodium chloride and 0.24% potassium dihydrogen phosphate.

[0038] Preferably, the freeze-drying procedure in step S2-8 is:

[0039] 1) Freezing: 4°C, 2h, -20°C, 2h, -60°C, 2h;

[0040] 2) Sublimation drying: set the vacuum degree to 5 and dry for 12 hours;

[0041] 3) Desorption and drying: Set the vacuum degree to 10 and dry for 12 hours.

[0042] The second aspect of the present invention provides a blood type reverse typing reagent, which is prepared by the method as described above, and the blood type reverse typing reagent includes type A red blood cell membrane antigen magnetic nanoparticles, type B red blood cell membrane antigen magnetic nanoparticles, and type O red blood cell membrane antigen magnetic nanoparticles.

[0043] The third aspect of the present invention provides a blood type reverse typing detection kit, which includes the blood type reverse typing reagent as described above.

[0044] The beneficial effects of the present invention are:

[0045] The present invention provides a blood type reverse typing reagent, a preparation method thereof and a blood type reverse typing detection kit. The present invention utilizes the advantages of excellent magnetic properties and biocompatibility of magnetosomes and surface display technology, expresses protein G on the surface of magnetosomes through genetic engineering and specifically binds to anti-H blood type antigen monoclonal antibodies, couples red blood cell membrane antigens to the surface of magnetosomes, and forms red blood cell membrane antigen magnetic nanoparticles, which can avoid the problem of easy hemolysis of red blood cells and disappearance of red blood cell membrane antigens under long-term storage. The magnetosomes are not only carriers of antigens, but also indicators of agglutination results, which facilitates result interpretation. In addition, the present invention uses magnetic separation technology to replace the centrifugal force in traditional methods, which can make detection more convenient and the required instruments more compact and portable; by preparing red blood cell membrane antigen magnetic nanoparticles and freeze-drying them, long-term stable storage of reverse typing red blood cell antigens can be achieved, which is conducive to the standardization and normalization of reverse typing red blood cell antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a flow chart for the preparation of red blood cell membrane antigen magnetic nanoparticles in Example 1 of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0048] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0049] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0050] Example 1

[0051] A method for preparing a blood type reverse typing reagent, characterized in that it comprises the following steps:

[0052] S1. Construction of functionalized magnetic nanoparticles expressing recombinant protein G:

[0053] S1-1. Construction of a magnetotactic bacterial MSR-1 mutant strain lacking bacterial magnetic particle membrane protein mamC / mamF (indicating mamC or mamF):

[0054] S1-1-1, amplify two homologous fragments of 700-1000 bp in length on both sides of the bacterial magnetic particle membrane protein gene; both homologous fragments have two restriction sites;

[0055] S1-1-2, the left and right homologous fragments were double-digested with the corresponding restriction endonucleases, and the suicide plasmid pK18mobsacB was double-digested at the same time;

[0056] S1-1-3, connect the left and right homologous fragments after double enzyme digestion and the suicide plasmid after double enzyme digestion, transfer the connection products into E. coli competent cells, and select the positive clones with correct connection;

[0057] S1-1-4. The positive cloned bacteria were used as donors for parental conjugation with the magnetotactic bacteria MSR-1 recipient bacteria, and the pK18mobsacB recombinant plasmid was introduced. The homologous integration mutants of the magnetotactic bacteria MSR-1 were screened by kanamycin resistance, and then the double exchange mutants of the magnetotactic bacteria MSR-1 were screened by sucrose gradient. Finally, the double exchange strains sensitive to kanamycin resistance were selected for verification, and the magnetotactic bacteria MSR-1 mutant strains with the bacterial magnetic particle membrane protein mamC / mamF gene missing were constructed.

[0058] S1-2. Construction of functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G:

[0059] S1-2-1, amplify the DNA fragment of the transmembrane domain of the bacterial magnetic particle membrane protein mamC / mamF gene, the two ends of the DNA fragment respectively have double enzyme cutting sites; perform double enzyme cutting on the amplified product DNA fragment and the pBBR plasmid, recover the double enzyme cutting DNA fragment and the double enzyme cutting pBBR plasmid; connect the double enzyme cutting DNA fragment with the double enzyme cutting pBBR plasmid, transform the connection product into Escherichia coli competent cells, screen the positive clones with correct connection, and obtain the recombinant plasmid pBBR-bacterial magnetic particle membrane protein mamC / mamF gene;

[0060] S1-2-2, clone the coding sequence of recombinant protein G into the pUC57 vector, and add double restriction sites at both ends;

[0061] S1-2-3, amplifying and double-enzyme digesting the recombinant protein G plasmid obtained by gene synthesis in step S1-2-2;

[0062] S1-2-4. The recombinant plasmid pBBR-bacterial magnetic particle membrane protein mamC / mamF gene obtained in step S1-2-1 is double-digested and purified, and connected with the gene fragment of the double-digested polypeptide chain-specific recombinant protein plasmid obtained in step S1-2-3. The connection product is transformed into Escherichia coli competent cells, and the positive clones with correct connection are screened and verified to finally obtain the functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR-bacterial magnetic particle membrane protein gene-recombinant protein G.

[0063] S1-3, the functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G is transferred into the magnetotactic bacteria MSR-1 mutant strain with bacterial magnetic particle membrane protein mamC / mamF gene deletion obtained in step S1 by conjugation, to construct the magnetotactic bacteria MSR-1 recombinant strain, and culture it to prepare the functional bacterial magnetic particles:

[0064] S1-3-1, using the functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G as a donor, it was introduced into the magnetotactic bacteria MSR-1 mutant strain lacking the bacterial magnetic particle membrane protein mamC / mamF gene through a parental conjugation experiment to construct a magnetotactic bacteria MSR-1 recombinant strain;

[0065] S1-3-2. The recombinant strain of magnetotactic bacteria MSR-1 is transferred and fermented, and the functional bacterial magnetic particles prepared by bacterial expression are determined by a magnetic device. The bacteria are collected by centrifugation and extracted and purified to obtain functional bacterial magnetic particles.

[0066] S2. Preparation of red blood cell membrane antigen magnetic nanoparticles:

[0067] Reference Figure 1 The main steps are: adding anti-H blood type monoclonal antibody to red blood cells, washing and lysing the red blood cells, then adding the functionalized magnetic nanoparticles prepared in step S1, washing, magnetically separating the products, and freeze-drying to obtain red blood cell membrane antigen magnetic nanoparticles, i.e., blood type reverse typing reagent; the specific steps are as follows:

[0068] S2-1. Collect fresh red blood cells, wash them three times with 0.9% NaCl solution (normal saline), and discard the supernatant;

[0069] S2-2, add anti-H blood group monoclonal antibody, incubate at 37°C for 30 minutes, wash three times with 0.9% NaCl solution, and discard the supernatant;

[0070] S2-3, adding a 0.6% NaCl solution of the same volume as the packed red blood cells (i.e., the red blood cell precipitate after the last centrifugation and discarding the supernatant in step S2-2), mixing and keeping at room temperature for 5 minutes, centrifuging at 2000 rpm for 5 minutes and collecting the precipitate;

[0071] S2-4, adding a 0.2% NaCl solution of the same volume as the packed red blood cells to the precipitate obtained in step S2-3, mixing and keeping at room temperature for 5 minutes, centrifuging at 3000 rpm for 10 minutes and collecting the precipitate;

[0072] S2-5, adding distilled water of the same volume as the packed red blood cells to the precipitate obtained in step S2-4, mixing and keeping at room temperature for 5 minutes;

[0073] S2-6, adding the functionalized magnetic nanoparticles prepared in step S1-1, and incubating at 37° C. for 30 minutes;

[0074] S2-7, magnetic adsorption, the magnetic product was washed three times with PBS;

[0075] S2-8. Add lyophilization protection solution to the product of step S2-7 and resuspend it. After packaging, freeze-dry it in a freeze dryer to obtain red blood cell membrane antigen magnetic nanoparticles, i.e., blood group reverse typing reagent, and store it at 2-8°C.

[0076] The freeze-dried protective solution includes the following components by mass concentration: 5% sucrose, 1% PEG4000, 1% bovine serum albumin, 3.58% disodium hydrogen phosphate dodecahydrate, 0.201% potassium chloride, 8.19% sodium chloride and 0.24% potassium dihydrogen phosphate.

[0077] Wherein, the freeze-drying procedure in step S2-8 is:

[0078] 1) Freezing: 4°C, 2h, -20°C, 2h, -60°C, 2h;

[0079] 2) Sublimation drying: set the vacuum degree to 5 and dry for 12 hours;

[0080] 3) Desorption and drying: Set the vacuum degree to 10 and dry for 12 hours.

[0081] Test Example 1 Red blood cell reverse typing test

[0082] Use type A red blood cells, type B red blood cells, and type O red blood cells of known blood type to prepare blood type reverse typing reagents in the manner of Example 1: type A red blood cell membrane antigen magnetic nanoparticles, type B red blood cell membrane antigen magnetic nanoparticles, and type O red blood cell membrane antigen magnetic nanoparticles to replace fresh red blood cells for ABO blood type system reverse typing detection.

[0083] The detection steps are as follows:

[0084] (1) Take one bottle of type A erythrocyte membrane antigen magnetic nanoparticles, one bottle of type B erythrocyte membrane antigen magnetic nanoparticles, and one bottle of type O erythrocyte membrane antigen magnetic nanoparticles, and add 500 μL of pure water to reconstitute them respectively;

[0085] (2) Take three clean test tubes and label them A, B, and O respectively;

[0086] (3) Add 50 μL of the sample to be tested (serum or plasma);

[0087] (4) Add 50 μL of the reconstituted A-type red blood cell membrane antigen magnetic nanoparticles, B-type red blood cell membrane antigen magnetic nanoparticles, and O-type red blood cell membrane antigen magnetic nanoparticles to the corresponding tubes respectively;

[0088] (5) Mix, magnetically adsorb, oscillate the test tube, and determine the result.

[0089] If the red blood cell membrane antigen magnetic nanoparticles agglutinate, it is a positive reaction; conversely, if the red blood cell membrane antigen magnetic nanoparticles do not agglutinate, it is a negative reaction.

[0090] The reaction criteria are as follows:

[0091]

[0092] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing a blood type reverse typing reagent, characterized in that: The following steps are involved: S1. Construction of functionalized magnetic nanoparticles expressing recombinant protein G; S2. Preparation of red blood cell membrane antigen magnetic nanoparticles: Anti-H blood type monoclonal antibody is added to the red blood cells, the red blood cells are lysed after washing, and then the functionalized magnetic nanoparticles prepared in step S1 are added, washed, magnetically adsorbed, and the magnetic product is freeze-dried to obtain red blood cell membrane antigen magnetic nanoparticles, i.e. the blood type reverse typing reagent.

2. The method for preparing the blood type reverse typing reagent according to claim 1, characterized in that: Step S1 specifically includes: S1-1, construction of a mutant strain of magnetotactic bacteria MSR-1 lacking the bacterial magnetic particle membrane proteins mamC / mamF; S1-2, construct functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G; S1-3. The functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G is transferred into the magnetotactic bacteria MSR-1 mutant strain lacking the bacterial magnetic particle membrane protein mamC / mamF gene obtained in step S1 through conjugation to construct a magnetotactic bacteria MSR-1 recombinant strain, culture it, and prepare functional bacterial magnetic particles.

3. The method for preparing the blood type reverse typing reagent according to claim 2, characterized in that: Step S1-1 specifically includes: S1-1-1, amplify two homologous fragments of 700-1000 bp in length on both sides of the bacterial magnetic particle membrane protein gene; both homologous fragments have two restriction sites; S1-1-2, the left and right homologous fragments were double-digested with the corresponding restriction endonucleases, and the suicide plasmid pK18mobsacB was double-digested at the same time; S1-1-3, connect the left and right homologous fragments after double enzyme digestion and the suicide plasmid after double enzyme digestion, transfer the connection products into E. coli competent cells, and select the positive clones with correct connection; S1-1-4. The positive cloned bacteria were used as donors for parental conjugation with the magnetotactic bacteria MSR-1 recipient bacteria, and the pK18mobsacB recombinant plasmid was introduced. The homologous integration mutants of the magnetotactic bacteria MSR-1 were screened by kanamycin resistance, and then the double exchange mutants of the magnetotactic bacteria MSR-1 were screened by sucrose gradient. Finally, the double exchange strains sensitive to kanamycin resistance were selected for verification, and the magnetotactic bacteria MSR-1 mutant strains with the bacterial magnetic particle membrane protein mamC / mamF gene missing were constructed.

4. The method for preparing the blood type reverse typing reagent according to claim 2, characterized in that: Step S1-2 specifically includes: S1-2-1, amplify the DNA fragment of the transmembrane domain of the bacterial magnetic particle membrane protein mamC / mamF gene, the two ends of the DNA fragment respectively have double enzyme cutting sites; perform double enzyme cutting on the amplified product DNA fragment and the pBBR plasmid, recover the double enzyme cutting DNA fragment and the double enzyme cutting pBBR plasmid; connect the double enzyme cutting DNA fragment with the double enzyme cutting pBBR plasmid, transform the connection product into Escherichia coli competent cells, screen the positive clones with correct connection, and obtain the recombinant plasmid pBBR-bacterial magnetic particle membrane protein mamC / mamF gene; S1-2-2, clone the coding sequence of recombinant protein G into the pUC57 vector, and add double restriction sites at both ends; S1-2-3, amplifying and double-enzyme digesting the recombinant protein G plasmid obtained by gene synthesis in step S1-2-2; S1-2-4. The recombinant plasmid pBBR-bacterial magnetic particle membrane protein mamC / mamF gene obtained in step S1-2-1 is double-digested and purified, and connected with the gene fragment of the double-digested polypeptide chain-specific recombinant protein plasmid obtained in step S1-2-3. The connection product is transformed into Escherichia coli competent cells, and the positive clones with correct connection are screened and verified to finally obtain the functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR-bacterial magnetic particle membrane protein gene-recombinant protein G.

5. The method for preparing the blood type reverse typing reagent according to claim 2, characterized in that: Step S1-3 specifically includes: S1-3-1, using the functional bacterial magnetic particle membrane protein fusion expression plasmid pBBR--bacterial magnetic particle membrane protein gene-recombinant protein G as a donor, it was introduced into the magnetotactic bacteria MSR-1 mutant strain lacking the bacterial magnetic particle membrane protein mamC / mamF gene through a parental conjugation experiment to construct a magnetotactic bacteria MSR-1 recombinant strain; S1-3-2. The recombinant strain of magnetotactic bacteria MSR-1 is transferred and fermented, and the functional bacterial magnetic particles prepared by bacterial expression are determined by a magnetic device. The bacteria are collected by centrifugation and extracted and purified to obtain functional bacterial magnetic particles.

6. The method for preparing the blood type reverse typing reagent according to claim 1, characterized in that: Step S2 specifically includes: S2-1. Collect fresh red blood cells, wash them three times with 0.9% NaCl solution, and discard the supernatant; S2-2, add IgG anti-H blood group monoclonal antibody, incubate at 37°C for 30 minutes, wash three times with 0.9% NaCl solution, and discard the supernatant; S2-3, add a 0.6% NaCl solution of equal volume to the packed red blood cells, mix well, keep at room temperature for 5 minutes, centrifuge at 2000 rpm for 5 minutes and collect the precipitate; S2-4, adding a 0.2% NaCl solution of the same volume as the packed red blood cells to the precipitate obtained in step S2-3, mixing and keeping at room temperature for 5 minutes, centrifuging at 3000 rpm for 10 minutes and collecting the precipitate; S2-5, adding distilled water of the same volume as the packed red blood cells to the precipitate obtained in step S2-4, mixing and keeping at room temperature for 5 minutes; S2-6, adding the functionalized magnetic nanoparticles prepared in step S1-1, and incubating at 37° C. for 30 minutes; S2-7, magnetic adsorption, the magnetic product was washed three times with PBS; S2-8, adding lyophilization protection solution to the product of step S2-7, resuspending, packaging and lyophilizing to obtain red blood cell membrane antigen magnetic nanoparticles, i.e., the blood group reverse typing reagent; Among them, red blood cells include type A red blood cells, type B red blood cells, and type O red blood cells, and the corresponding prepared red blood cell membrane antigen magnetic nanoparticles are type A red blood cell membrane antigen magnetic nanoparticles, type B red blood cell membrane antigen magnetic nanoparticles, and type O red blood cell membrane antigen magnetic nanoparticles.

7. The method for preparing the blood type reverse typing reagent according to claim 6, characterized in that: The freeze-dried protective solution includes the following components by mass concentration: 5% sucrose, 1% PEG4000, 1% bovine serum albumin, 3.58% disodium hydrogen phosphate dodecahydrate, 0.201% potassium chloride, 8.19% sodium chloride and 0.24% potassium dihydrogen phosphate.

8. The method for preparing the blood type reverse typing reagent according to claim 6, characterized in that: The freeze-drying procedure in step S2-8 is: 1) Freezing: 4°C, 2h, -20°C, 2h, -60°C, 2h; 2) Sublimation drying: set the vacuum degree to 5 and dry for 12 hours; 3) Drying: Set the vacuum degree to 10 and dry for 12 hours.

9. A blood type reverse typing reagent, characterized in that: It is prepared by the method according to any one of claims 1 to 8, and the blood type reverse typing reagent comprises type A red blood cell membrane antigen magnetic nanoparticles, type B red blood cell membrane antigen magnetic nanoparticles, and type O red blood cell membrane antigen magnetic nanoparticles.

10. A blood type reverse typing detection kit, characterized in that: It comprises the blood group reverse typing reagent as claimed in claim 9.

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