Two-strain anti-newcastle disease virus nano antibody and expression preparation method and application thereof
阅读说明：本技术 两株抗新城疫病毒纳米抗体及其表达制备方法和应用 (Two-strain anti-newcastle disease virus nano antibody and expression preparation method and application thereof ) 是由 赵钦 孙亚妮 纪品品 周恩民 于 2019-10-30 设计创作，主要内容包括：本发明公开了一种抗新城疫病毒的纳米抗体NDV-Nb4和NDV-Nb49,氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示。还公开了由纳米抗体NDV-Nb4与铁蛋白融合构建融合蛋白的方法,通过原核表达系统可溶性表达纳米抗体与铁蛋白的融合蛋白；以及由纳米抗体NDV-Nb49与辣根过氧化物酶融合构建融合蛋白的方法,通过真核表达系统表达NDV-Nb49与辣根过氧化物酶融合蛋白。还公开了一种检测新城疫病毒的方法,是以NDV-Fe-Nb4融合蛋白为捕获NDV的蛋白、NDV-Nb49-HRP融合蛋白为检测NDV的蛋白,配合用于检测NDV,该方法简便易操作,且特异性好,准确率高。(The invention discloses nano antibodies NDV-Nb4 and NDV-Nb49 for resisting newcastle disease virus, wherein amino acid sequences are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Also discloses a method for constructing fusion protein by fusing nano antibody NDV-Nb4 and ferritin, wherein the fusion protein of the nano antibody and ferritin is soluble expressed by a prokaryotic expression system; and a method for constructing fusion protein by fusing nano antibody NDV-Nb49 and horseradish peroxidase, and expressing the fusion protein of NDV-Nb49 and horseradish peroxidase by a eukaryotic expression system. The NDV-Fe-Nb4 fusion protein is used as a protein for capturing NDV, and the NDV-Nb49-HRP fusion protein is used as a protein for detecting NDV, and is matched for detecting NDV.)
1. A nanometer antibody NDV-Nb4 for resisting Newcastle disease virus is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown.
2. A nanometer antibody NDV-Nb49 for resisting Newcastle disease virus is characterized in that the amino acid sequence is shown as SEQ ID NO: 2, respectively.
3. A method for constructing a fusion protein by fusing the nano antibody NDV-Nb4 against Newcastle disease virus and ferritin according to claim 1, which comprises the following steps:
step 1: designing primers NDV-4-FeNb-Nde I-F and NDV-4-Ferritin-R according to the nucleotide sequence of the encoded Ferritin, and obtaining the nucleotide sequence of the Ferritin by PCR amplification by taking an encoded Ferritin recombinant plasmid pUC-57-Fe as a template;
step 2: designing primers NDV-4-Nb-F and NDV-4-FeNb-BamH I-R according to the nucleotide sequence of the nano antibody NVD-Nb4 for encoding the Newcastle disease virus, and carrying out PCR amplification on the recombinant plasmid pMERC-NDV-Nb4 containing the NDV-Nb4 gene obtained in the step 1 to obtain a nano antibody sequence;
and step 3: taking the two target gene fragments obtained in the step 1 and the step 2 as templates, and carrying out PCR amplification by using an upstream primer NDV-4-FeNb-Nde I-F for amplifying the ferritin gene and a downstream primer NDV-4-FeNb-BamH I-R for amplifying the nano antibody NDV-Nb4 gene to obtain an NDV-Fe-Nb4 fusion gene;
and 4, step 4: and performing double enzyme digestion on the obtained NDV-Fe-Nb4 fusion gene and a prokaryotic expression vector pET-28a, connecting, then transferring into competent escherichia coli to obtain a positive recombinant plasmid pET-28a-NDV-Fe-Nb4, converting the obtained positive plasmid pET-28a-NDV-Fe-Nb4 into the competent escherichia coli, and performing separation and purification after culture to obtain the NDV-Fe-Nb4 fusion protein.
4. The method for constructing fusion protein by fusing nano antibody NDV-Nb4 against Newcastle disease virus with ferritin according to claim 3, wherein the primer NDV-4-FeNb-Nde I-F in step 1 is shown in SEQ ID NO: 3 is shown in the specification;
the primer NDV-4-Ferritin-R is shown as SEQ ID NO: 4, respectively.
5. The method for constructing fusion protein by fusing nano antibody NDV-Nb4 against Newcastle disease virus and ferritin according to claim 3, wherein the primer NDV-4-Nb-F in step 2 is shown in SEQ ID NO: 5 is shown in the specification;
the primer NDV-4-FeNb-BamH I-R is shown as SEQ ID NO: and 6.
6. A method for constructing a fusion protein by fusing the nano antibody NDV-Nb49 against Newcastle disease virus and horseradish peroxidase according to claim 2, which is characterized by comprising the following steps:
step A: carrying out double enzyme digestion on the NDV-Nb49 fragment and the pCMV-HRP vector respectively, then connecting the fragments by using ligase, transferring the connecting product into competent escherichia coli, and coating an LB (Langmuir-Blodgett) plate to obtain a positive plasmid;
and B: transferring the obtained positive plasmid into HET293T cells, collecting cell secretion expression supernatant, and purifying to obtain NDV-Nb49-HRP fusion protein.
7. The method for constructing a fusion protein according to claim 6, wherein the NDV-Nb49-HRP fusion protein is obtained by purifying the supernatant in step B by SDS-PAGE, performing electrotransformation on a nitrocellulose membrane, and identifying the protein by Western blotting analysis.
8. An NDV-Fe-Nb4 fusion protein obtained by the method for constructing a fusion protein according to any one of claims 3 to 5.
9. An NDV-Nb49-HRP fusion protein obtained by the method of constructing a fusion protein according to any one of claims 6 to 7.
10. A method for detecting Newcastle disease virus, which comprises using the NDV-Fe-Nb4 fusion protein of claim 8 as a protein for capturing NDV and the NDV-Nb49-HRP fusion protein of claim 9 as a protein for detecting NDV, in combination for detecting NDV.
Technical Field
The invention relates to the technical field of biology, in particular to two strains of Newcastle disease virus resistant nano antibodies and an expression preparation method and application thereof.
Background
The nano antibody is a heavy chain antibody which is naturally deleted of a light chain and is found in camel blood by Hamers-Casterman et al in 1989, and has the molecular weight of 15kDa, the diameter of 2.2nm and the length of 4.8 nm. Compared with the traditional antibody, the nano antibody has the advantages of higher affinity, higher water solubility, stable conformation, easy genetic engineering modification, blood brain barrier crossing and the like. In recent years, with the research on nano antibodies, the antibodies have been widely used in the fields of protein visualization tracing, structure analysis, diagnosis and treatment of epidemic diseases of human beings and animals, and the like. In the existing immunological diagnostic techniques for pathogens, antibodies play a significant role. For example, in the ELISA detection method of pathogenic antibody, the production of secondary antibody or anti-pathogenic antibody determines the production cost and market promotion and application value of the detection method. However, the production of conventional antibodies requires animal or fermentation cell culture, resulting in high production cost and complicated production process. The nano antibody has the advantages of easy genetic engineering modification, low production cost and simple production process. Therefore, the antibody can replace the traditional antibody, is applied to the research and development of the immunological diagnosis technology of pathogeny, and has wide market application prospect.
Newcastle Disease (ND) is an acute, highly contagious disease mainly infecting birds caused by Newcastle Disease Virus (NDV). The disease is mainly characterized by digestive tract and respiratory tract pathological changes, has high morbidity and mortality, and brings serious harm to poultry industry. In China, NDV inactivation and attenuated vaccines are widely used, so that outbreak and prevalence of ND are well controlled. However, the toxicity of different strains of NDV is greatly different, and the virus also has certain variation along with the time under high immune pressure, which brings difficulty to NDV detection work.
Currently, the NDV detection methods mainly include separation and identification of viruses, electron microscopy detection techniques, serological diagnosis methods, molecular biological diagnosis methods, and the like. The ELISA method is a main method for diagnosing and detecting animal epidemic diseases due to simple operation and high flux. ELISA methods are various, including direct ELISA, indirect ELISA, double antibody sandwich ELISA, etc. Among them, the double antibody sandwich ELISA method is widely used in the detection of pathogens, and many commercial kits have been produced due to the advantages of high flux, high sensitivity and strong specificity. A method for establishing a pathogenic double-antibody sandwich ELISA detection method comprises the steps of firstly, screening and preparing two paired antibodies (a capture antibody and a detection antibody), wherein the capture antibody needs strong antigen capturing capacity, and the detection antibody needs to be marked by HRP or other enzymes. Therefore, in the preparation of commercial kits, a large amount of antibody is required to be prepared and labeled. However, due to the problems of enzyme labeling efficiency and antibody production process, the production cost of the kit is high, and the kit is difficult to popularize and use clinically or in a basic level on a large scale. The nano antibody has the characteristics of small molecular weight and easy genetic engineering modification. Therefore, the sandwich ELISA method is established by replacing the traditional antibody with the nano antibody, and the production process is simplified and the production cost is reduced by modifying the nano antibody, so that the sandwich ELISA method has important significance.
Disclosure of Invention
The invention aims to provide nano antibodies NDV-Nb4 and NDV-Nb49 for resisting Newcastle disease virus. Meanwhile, a method for constructing fusion protein by fusing nano antibody NDV-Nb4 of anti-Newcastle disease virus and ferritin is also provided, and the fusion protein of the nano antibody and the ferritin is soluble expressed by constructing a prokaryotic expression system; the fusion protein is constructed by fusing nano antibody NDV-Nb49 of anti-Newcastle disease virus and horseradish peroxidase, and the fusion protein of NDV-Nb49 and horseradish peroxidase is expressed by utilizing a eukaryotic expression system HEK293T cell.
The invention also aims to provide a method for detecting Newcastle disease virus, and particularly, the NDV-Fe-Nb4 fusion protein is used as a protein for capturing NDV, and the NDV-Nb49-HRP fusion protein is used as a protein for detecting NDV, and is matched for detecting NDV.
To achieve these objects and other advantages of the present invention, there is provided a nanobody NDV-Nb4 against newcastle disease virus, having an amino acid sequence as set forth in SEQ ID NO: 1 is shown.
The invention also provides a nano antibody NDV-Nb49 for resisting the Newcastle disease virus, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.
The invention also provides a method for constructing fusion protein by fusing the nano antibody NDV-Nb4 for resisting the Newcastle disease virus and ferritin, which comprises the following steps:
step 1: designing primers NDV-4-FeNb-Nde I-F and NDV-4-Ferritin-R according to the nucleotide sequence of the encoded Ferritin, and obtaining the nucleotide sequence of the Ferritin by PCR amplification by taking an encoded Ferritin recombinant plasmid pUC-57-Fe as a template;
step 2: designing primers NDV-4-Nb-F and NDV-4-FeNb-BamH I-R according to the nucleotide sequence of the nano antibody NVD-Nb4 for encoding the Newcastle disease virus, and carrying out PCR amplification on the recombinant plasmid pMERC-NDV-Nb4 containing the NDV-Nb4 gene obtained in the
and step 3: taking the two target gene fragments obtained in the
and 4, step 4: and performing double enzyme digestion on the obtained NDV-Fe-Nb4 fusion gene and a prokaryotic expression vector pET-28a, connecting, then transferring into competent escherichia coli to obtain a positive recombinant plasmid pET-28a-NDV-Fe-Nb4, converting the obtained positive plasmid pET-28a-NDV-Fe-Nb4 into the competent escherichia coli, and performing separation and purification after culture to obtain the NDV-Fe-Nb4 fusion protein.
Preferably, the primer NDV-4-FeNb-Nde I-F in
the primer NDV-4-Ferritin-R is shown as SEQ ID NO: 4, respectively.
Preferably, the primer NDV-4-Nb-F in
the primer NDV-4-FeNb-BamH I-R is shown as SEQ ID NO: and 6.
The invention also provides a method for constructing fusion protein by fusing the nano antibody NDV-Nb49 for resisting the Newcastle disease virus and horseradish peroxidase, which comprises the following steps:
step A: carrying out double enzyme digestion on the NDV-Nb49 fragment and the pCMV-HRP vector respectively, then connecting the fragments by using ligase, transferring the connecting product into competent escherichia coli, and coating an LB (Langmuir-Blodgett) plate to obtain a positive plasmid;
and B: transferring the obtained positive plasmid into HET293T cells, collecting cell secretion expression supernatant, and purifying to obtain NDV-Nb49-HRP fusion protein.
Preferably, in the step B, after the supernatant is purified by SDS-PAGE, after the supernatant is electrically transformed to a nitrocellulose membrane, the NDV-Nb49-HRP fusion protein is obtained by Western blotting analysis and identification.
The invention also provides the NDV-Fe-Nb4 fusion protein obtained by the method for constructing the fusion protein.
The invention also provides the NDV-Nb49-HRP fusion protein obtained by the method for constructing the fusion protein.
The invention also provides a method for detecting the Newcastle disease virus, which takes the NDV-Fe-Nb4 fusion protein as a protein for capturing NDV, and takes the NDV-Nb49-HRP fusion protein as a protein for detecting NDV, and is matched with the protein for detecting NDV.
The invention discloses the following technical effects:
the invention provides a nano antibody NDV-Nb4 for resisting Newcastle disease virus, and by constructing a fusion expression vector of the NDV-Nb4 nano antibody and ferritin, a prokaryotic expression system is used for expressing the fusion protein, and the fusion protein is used as a capture antibody, so that the capacity of capturing the Newcastle disease virus can be greatly improved. Meanwhile, another NDV-Nb49 nano antibody and HRP fusion expression vector is constructed, the fusion protein is expressed by utilizing a eukaryotic expression system, and the protein is used as a detection antibody and can well detect the trapped Newcastle disease virus. The two strains of the nano antibody fusion protein are used in a matched mode, so that the antigen capture capacity is greatly improved, the antibody does not need to be purified and marked, the antibody can be directly applied to the establishment of an ELISA method, the production process is greatly simplified, and the production cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is an electrophoretogram constructed by the NDV-Nb-4 gene and ferritin fusion recombinant expression vector of the present invention; wherein, a: respectively amplifying a gene sequence of ferritin and a gene sequence of NDV-Nb-4 by PCR, wherein M: nucleic acid Maker; 1: amplifying a ferritin gene; 2: amplifying the gene of NDV-Nb-4; b: carrying out PCR amplification on a fusion gene segment of NDV-Fe-Nb4 and ferritin; c: double enzyme digestion recombinant vector and fusion gene fragment, M: nucleic acid Maker; 1: cutting the recombinant vector pET28a into fragments after enzyme digestion; 2: fusing gene segments after enzyme digestion; d: and (3) reconstructing a bacterial liquid PCR electrophoresis picture of the NDV-capture antibody, wherein M: nucleic acid Maker; NC: negative control;
FIG. 2 shows the results of prokaryotic expression and purification of a capture antibody fused between an NDV nanobody and ferritin according to the present invention; wherein, a: SDS-PAGE analysis for prokaryotic expression of NDV nanobody fused to ferritin capture antibody, M: protein Maker; lane 1: inducing and expressing thallus lysate; lane 2: supernatant after ultrasonic treatment; lane 3: inclusion bodies; b: purification of NDV Nanobody fusion with ferritin capture antibody, M: protein Maker; lane 1: supernatant after ultrasonic treatment; lane 2: discharging liquid; lane 3-5: 10mM imidazole eluting heteroprotein; lane 6-14: eluting the target protein; c: analyzing prokaryotic expression of a capture antibody fused by the NDV nano antibody and ferritin by Western blot; d: observing self-assembly of the NDV nano antibody and the ferritin fused capture antibody by an electron microscope;
FIG. 3 shows the results of the present ELISA to verify the specific binding and capture of NDV-Fe-
FIG. 4 shows the results of eukaryotic expression and purification of an antibody for detecting HRP fused with an NDV nanobody according to the present invention; wherein, a: double enzyme digestion recombinant vector and fusion gene segment; b: reconstructing a bacterial liquid PCR electrophoresis chart of a carrier NDV-detection antibody; lanes 1-5 different bacterial clones; c: IFA analysis NDV nanometer antibody fuses the eukaryotic expression of detection antibody of HRP; d: analyzing the eukaryotic expression of a detection antibody fused by the NDV nano antibody and the HRP by Western blot;
FIG. 5 shows the results of the present ELISA verifying that NDV-Nb49-HRP cell secretion supernatant specifically binds to NDV virus particles; wherein, a: specific binding of NDV-Nb49-HRP to NDV virus particles; b: NDV-Nb49-HRP cell secretion supernatants at different dilutions were combined with NDV virions;
FIG. 6 shows the detection of the change in different dilutions of NDV virus particles according to the present invention; wherein, a: detecting different chick embryo allantoic fluid NDV virus particle dilution gradients; b: detecting different masses of purified NDV viral particles; c: linear regression equation of chick embryo allantoic fluid NDV virus particles with different hemagglutination titers; d: linear regression equations for different masses of purified NDV viral particles;
FIG. 7 shows the result of the specificity analysis of the double antibody sandwich ELISA of the present invention.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
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