CN108285903B - Preparation method of specific stimulating antigen - Google Patents
Preparation method of specific stimulating antigen Download PDFInfo
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- CN108285903B CN108285903B CN201810084733.6A CN201810084733A CN108285903B CN 108285903 B CN108285903 B CN 108285903B CN 201810084733 A CN201810084733 A CN 201810084733A CN 108285903 B CN108285903 B CN 108285903B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention relates to a preparation method of a specific stimulating antigen, which uses cross-species protein as the specific stimulating antigen, in particular to a preparation method of a specific stimulating antigen of a human antibody by using zebrafish protein by utilizing the characteristic of TGF-beta antibody cross-species reaction. The cross-species protein preparation method comprises the following steps: optimizing the whole gene sequence of the zebra fish inhibin B; designing a full-length splicing primer; constructing a recombinant plasmid; the inhibin B protein is prepared by transfecting host cells and is used as an antigen for preparing a specific antibody of human inhibin B. The invention adopts a method of taking cross-species protein as a specific stimulating antigen and takes artificially synthesized cross-species inhibin B protein as an antigen to carry out an immune test, thereby obtaining the inhibin B antibody with high purity and providing a new immune strategy for obtaining the human specific antibody.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a preparation method of a specific stimulating antigen.
Background
Inhibin is one of the members of the transforming growth factor beta (TGF-beta) superfamily, a dimeric glycoprotein hormone consisting of two subunits, alpha and beta. The relative molecular mass of the alpha subunit (Mr) is 20000; the relative molecular mass (Mr) of the beta subunit is 5000-. Alpha and beta are combined through disulfide bonds to form inhibin A (alpha beta A) and inhibin B (alpha beta B), and the other two beta subunits can also form activin A (beta A), activin B (beta B) and activin AB (beta A beta B). INH-B exists in organisms in a variety of molecular forms, including mature (Mr: 32000) and incompletely mature (also called precursors) α β dimer molecules and free α subunits. The alpha subunit preprotein (Pro-alpha C) in plasma can also be detected by enzyme-linked immunosorbent assay (ELISA), which is a possible preprotein series cross-reactive with other inhibins. The beta B subunit precursor is 1 peptide chain consisting of 392 amino acid (aa) residues, and the mature beta B subunit (115aa) is positioned at the carbon end of the peptide chain. The homology of nucleotide sequences of beta B subunit full-length coding regions of different mammals is more than 80%, the homology of nucleotide sequences of mature regions is more than 90%, and the homology of deduced amino acid sequences of mature regions is more than 96%. This sequence conservation of inhibin B among species, in turn, results in the property that antibodies directed against this molecule will have cross-species reactivity.
The subunit polymer structure of inhibin/activin, and different subunits have sequence mechanism similarity, so that when the respective antibodies are used for detection, the specificity of the antibodies for recognizing various naturally-occurring polymer antigens is greatly reduced, and therefore, the actual detection of various subunit polymer antigens has great cross reaction, which brings error on the result of protein detection, especially enzyme-linked immunosorbent assay (ELISA), and leads to the accuracy of the detection result to be not guaranteed, and the property of inhibin B molecule is particularly outstanding.
The most key to the preparation of the antibody is the selection of the antigen, the antigen source in the preparation of the INH-Bbeta subunit antibody is basically synthesized polypeptide, most of the antigen source is mature region C-terminal sequence, an animal (rabbit or mouse) is immunized by the synthesized polypeptide, a polyclonal antibody or a monoclonal antibody is prepared, the polypeptide is simultaneously used as a standard curve or/and a quality standard of a detection kit, but the detection target is natural inhibin B molecule, so the detection result is not accurate, and the detection result needs to be corrected by using a natural sample. The standard product can not be used for preparing a standard curve by a multiple dilution method like other protein detection methods, so that the preparation difficulty of the standard curve and the complexity of operation are improved.
The inhibin B complete gene sequence is synthesized by applying a genetic engineering technology to prepare cross-species recombinant protein which can be used as an antigen for preparing a human protein antibody, and the technical scheme for preparing a high-specificity antibody by using the cross-species protein as the antigen is realized.
Disclosure of Invention
Inhibin B belonging to TGF-beta superfamily has the characteristic of easily generating cross reaction with inhibin A and activin B in detection, and the main reason of the problem is that the prepared antibody can not specifically identify human inhibin B molecule with natural structure, and the antibody aiming at beta B chain of inhibin B molecule on the market also has cross reaction with VA chain and/or alpha chain. In order to overcome the defects of the prior art, the invention aims to solve the problem of a preparation method of a special antigen required by preparing an antibody with specific reaction.
To achieve the above object, the present invention adopts the following embodiments:
the characteristic that the subunits of inhibin B protein molecules have sequence similarity and high conservation of subunit sequences and the characteristic that an antibody of TGF-beta superfamily protein has strong cross-species reaction are utilized, and cross-species protein is taken as an antigen to prepare a high-specificity antibody. Zebra fish protein can be used as antigen for preparing humanized protein antibody. For example: in order to prepare an antibody having high specificity for the reaction with human inhibin B, zebrafish inhibin B protein was used as an antigen.
Specific embodiments of the present invention include:
a. cross-species proteins were used as specific stimulating antigens.
b. Zebrafish protein was used as a specific stimulating antigen for the preparation of human antibodies.
c. The zebra fish inhibin B protein is used as a characteristic stimulating antigen for preparing a human inhibin B antibody.
d. The zebrafish inhibin B protein beta chain is used as a characteristic stimulating antigen for preparing a human inhibin B antibody.
e. The zebrafish inhibin B protein beta chain is used as a characteristic stimulating antigen for preparing a human inhibin B beta chain antibody.
The preparation method of the zebra fish inhibin B protein antigen comprises the following steps:
(1) optimizing the whole gene sequence of the zebra fish inhibin B to obtain a target gene shown as SEQ NO. 1;
(2) designing a full-length splicing primer, and inserting the target gene synthesized in the step (1) into a vector plasmid through restriction endonuclease;
(3) transforming the plasmid containing the target gene in the step (2) into a host cell for expression;
(4) culturing the host cell transformed in the step (3) to obtain a fermentation liquid containing a large amount of recombinant protein;
(5) and (4) separating and purifying the fermentation liquor obtained in the step (4) to obtain the high-purity recombinant inhibin B protein.
Furthermore, the BALB/c mouse is immunized by the recombinant inhibin B protein prepared by the technical scheme of the invention to generate an anti-inhibin B monoclonal antibody, and the antibody protein with the purity of more than 90 percent is obtained by purifying ProteinA; meanwhile, the recombinant inhibin B protein can be used as an antigen for preparing a human protein antibody.
The invention has the beneficial effects that: the antibody of the TGF-beta superfamily protein has the characteristic of strong cross-species reaction, and the cross-species protein is used as an antigen to prepare a high-specificity antibody, thereby providing a new immunization strategy. The zebra fish recombinant inhibin B protein is used as an antigen for preparing a human source protein antibody, is used for preparing a human inhibin B antibody, has the purity of more than 90 percent, good activity and strong specificity, and can identify a human inhibin B molecule with a natural structure. In addition, the method is easy to realize the conversion of results from small research and development tests to industrial production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 is a diagram showing the double restriction sites NdeI and HindIII at both ends of the target sequence.
FIG. 2 shows the electrophoretogram of recombinant plasmid. Lane 1 was not digested and as a control, lane 2 was digested to show two bright bands, with a clear band at 1100 bp.
FIG. 3 shows the electrophoresis chart of the expression and purification of recombinant protein Inhibin, beta B.
FIG. 4 shows the antibody purity verification and WB gel electrophoresis.
Detailed Description
The technical solution of the present invention is described in detail below with reference to specific embodiments. It should be noted that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. Other embodiments, which can be derived by one of ordinary skill in the art from the embodiments of the present invention without creative efforts, are within the scope of the present invention.
Example 1 strategy using cross-species proteins as specific antigens.
By utilizing the characteristics of sequence similarity among subunits of inhibin B protein molecules and high conservation of subunit sequences and the characteristic of strong cross-species reaction of an antibody of TGF-beta superfamily protein, cross-species protein can be used as a specific antigen. Further, zebrafish protein can be used as a characteristic antigen for preparing human antibodies. Further, zebrafish inhibin B protein can be used as a characteristic antigen for preparing human inhibin B antibody. Further, the beta chain of the zebrafish inhibin B protein can be used as a characteristic antigen for preparing a human inhibin B antibody. Furthermore, the zebrafish inhibin B protein beta chain can be used as a characteristic antigen for preparing human inhibin B beta chain antibody.
Example 2 preparation of zebrafish inhibin B protein.
S1, optimizing the whole gene sequence of zebra fish inhibin B to obtain a target gene, which is shown in SEQ NO. 1;
s2 designs full-length splicing primers, inserts the target gene synthesized in S1 into a vector plasmid pET30a through restriction enzyme sites NdeI and Hind III, and transforms the target gene into BL21(DE3) host cells for expression;
s3 culturing the transformed host cell in S3 to obtain fermentation liquid containing a large amount of recombinant protein;
s4, the fermentation liquor obtained in the step S4 is separated and purified, and the recombinant inhibin B protein with high purity is obtained.
EXAMPLE 3 quality control of purified inhibin B protein
a. Inhibin B protein stability test (freeze-thaw experiment): and (3) taking one part of inhibin B protein which is frozen at-80 ℃ after being subpackaged, placing the part of inhibin B protein in an ice water mixture for slow melting, and having no abnormal phenomenon after melting, which indicates that the inhibin B protein freeze-thaw experiment is normal.
b. Determination of inhibin B protein concentration: protein concentration was determined using the Bradford protein concentration assay kit.
c. And (3) detecting WB of inhibin B protein: the WB experimental operation flow refers to Yao Jun treatise of protein electrophoresis Experimental technology. The detection results are shown in FIG. 3. As can be seen from FIG. 3, a clear band appears at 50kDa in SDS-PAGE of recombinant inhibin B protein, and a target band appears at the same position as detected by Western-Blot. The purity of the protein measured by a Western Blot method reaches over 90 percent.
EXAMPLE 4 inhibin B protein immunization of mice
The conventional antibody preparation method is adopted, and the recombinant inhibin B protein is used as an antigen to immunize a host BALB/c pure line mouse. And (3) carrying out ProteinA affinity purification on the anti-inhibin B monoclonal antibody generated by the immune mouse, carrying out SDS-PAGE electrophoresis on the purified antibody, staining by Coomassie brilliant blue, and detecting the purity of the antibody. The detection results are shown in FIG. 4. As can be seen in FIG. 4, the SDS-PAGE pattern of the monoclonal antibody shows a band of the antibody heavy chain at 55kD and a band of the antibody light chain at 25 kD. And detecting the occurrence of a target band at the same position by Western-BIot detection, and determining the purity of the antibody to be more than 90%.
Thus, the inhibin B protein of the inventive strategy can be used as an antigen of a cross-species bioprotein antibody. Further, zebrafish inhibin B protein can be used as a specific antigen for preparing human inhibin B antibody. Furthermore, the beta chain of the zebrafish inhibin B protein can be used as a characteristic antigen for preparing human inhibin B beta chain antibodies.
SEQUENCE LISTING
<110> institute of science and technology of the national institute of health and science and technology
<120> a method for preparing a specific stimulating antigen
<130> 2018.01.15
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1125
<212> DNA
<213> Artificial sequence of Zebra fish inhibin B
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cacccgattc cgaaagcagc aatggttacc gcactgcgta aactgcacgc aggtaaagtt 240
cgcgaagacg gtcgcgttga aattccgaat ctggatggtc acgcagcaca taacgaagtt 300
caggaagaaa ccagcgagat catcagcttt gcggaatctg acgacgttac cccgagtaaa 360
agcagtctgt acttcctgat cagcaacgaa ggcaaccaga acctgtacgt tctgcaagcg 420
aacctgtggc tgtacttcaa actgatgccg ggtaccctgg aaaaaggtct gcgtcgtaaa 480
gtcaccgttc gcgttcattc ttacgaaccg ggtggtcaaa acgttcactg gccgatgatg 540
gagaaacgcg ttgaactgaa acgtagcggt tggcatacct ttccggtttc tgaagccatt 600
cgcgaaatgc tggcaaaagg cggtcgtcgt caagatctgg atatccactg cgaaggttgc 660
gaagcagcaa acgttctgcc gattctggtt gatccgtctg atccgagtca tcgtccgttt 720
ctggttgttc gcgcacaaca agcagacggt aaacatcgta ttcgtaaacg cggtctggag 780
tgcgacggta ataacggggg tctgtgttgt cgtcagcagt tctacatcga ctttcgcctg 840
atcggttgga acgattggat cattgcaccg gcaggctatt acggcaacta ttgcgaaggc 900
agttgtccgg catatatggc aggcgttccg ggtagcgcaa gtagctttca taccgccgtt 960
gttaaccagt atcgtatgcg cggcatgagt ccgggttctg ttaatagctg ctgcattccg 1020
accaaactgt ccaccatgag catgctgtac ttcgacgacg agtacaacat cgtcaaacgc 1080
gacgttccga acatgatcgt cgaagagtgc ggttgcgcat aatga 1125
Claims (5)
1. A method for preparing a specific stimulating antigen, comprising: the zebrafish inhibin B protein beta chain is used as a characteristic stimulating antigen for preparing a human inhibin B antibody, and the sequence of the zebrafish inhibin B is SEQ NO. 1.
2. The method for producing a specific stimulating antigen according to claim 1, wherein: the zebrafish inhibin B protein beta chain is used as a characteristic stimulating antigen for preparing a human inhibin B beta chain antibody.
3. The method for preparing a specific stimulatory antigen of any one of claims 1-2, wherein the zebrafish inhibin B protein is prepared by:
s1, optimizing the whole gene sequence of zebra fish inhibin B to obtain a target gene SEQ NO. 1;
s2 designing a full-length splicing primer, constructing a recombinant plasmid and transfecting a host cell for expression;
s3, carrying out cell culture to obtain fermentation liquor containing a large amount of recombinant proteins;
s4 separating and purifying to obtain high-purity recombinant inhibin B protein.
4. The method for producing a specific stimulating antigen according to claim 3, wherein: the zebra fish inhibin B Protein is used as an antigen immune host BALB/c mouse to generate an anti-inhibin B monoclonal antibody, and the antibody Protein with the purity of more than 90 percent is obtained by purifying Protein A.
5. The method for producing a specific stimulating antigen according to claim 3, wherein: the zebra fish inhibin B protein is used as an antigen for preparing a human protein antibody.
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| NZ231899A (en) * | 1985-10-03 | 1991-07-26 | Genentech Inc | Human or porcine inhibin peptide compositions and dna encoding them |
| US8383351B2 (en) * | 2008-06-11 | 2013-02-26 | Oxford Brookes University | Antibody to inhibin/ activin β-B subunit |
| CN104730247A (en) * | 2015-03-12 | 2015-06-24 | 广州市丰华生物工程有限公司 | Kit suitable for rapidly detecting AMH and INHB by using double-tagging time resolution fluorescence immunoassay method and use method of kit |
| CN105527444B (en) * | 2015-12-21 | 2018-08-24 | 深圳华康生物医学工程有限公司 | The enzyme-linked immunologic detecting kit and inhibin B test methods of inhibin B |
| CN107024591A (en) * | 2017-05-02 | 2017-08-08 | 浙江星博生物科技股份有限公司 | Flow cytometer detection reagent for detecting InhibinB contents and its preparation method and application |
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