阅读说明：本技术 一种抗降钙素原的高亲和力纳米抗体及其应用 (Anti-procalcitonin high-affinity nano antibody and application thereof ) 是由 林景涛 宋海鹏 于建立 刘原源 于 2019-11-24 设计创作，主要内容包括：本发明公开了一种抗降钙素原的高亲和力纳米抗体,所述纳米抗体具有独特的3个互补决定区CDR1、CDR2、CDR3,本发明还提供了含有该纳米抗体可变区编码序列的表达载体,以及含有该表达载体的宿主细胞,本发明还提供了纳米抗体可变区与碱性磷酸酶的融合蛋白,所述纳米抗体在制备降钙素原检测试剂盒中的应用,应用所述纳米抗体进行降钙素原免疫检测的方法,以及相应的检测试剂盒。本发明提供的抗降钙素原纳米抗体对降钙素原具有特异的识别和结合能力,该纳米抗体亲和力可达到3.773E-9,具有独特的抗原决定簇识别位点,能够在降钙素原检测特别是双抗体夹心法中获得优异的检测效果。(The invention discloses a high-affinity nano antibody for resisting procalcitonin, which has 3 unique complementarity determining regions CDR1, CDR2 and CDR3, an expression vector containing a variable region coding sequence of the nano antibody, a host cell containing the expression vector, a fusion protein of a variable region of the nano antibody and alkaline phosphatase, an application of the nano antibody in preparing a procalcitonin detection kit, a method for performing procalcitonin immunodetection by using the nano antibody and a corresponding detection kit. The anti-procalcitonin nano antibody provided by the invention has specific recognition and binding capacity to procalcitonin, the affinity of the nano antibody can reach 3.773E-9, the nano antibody has a unique antigenic determinant recognition site, and an excellent detection effect can be obtained in procalcitonin detection, particularly in a double-antibody sandwich method.)

1. The high-affinity nanobody against procalcitonin is characterized in that the variable region of the nanobody has 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the sequence of the CDR1 region consists of the amino acid sequence shown in SEQ ID NO.1, the sequence of the CDR2 region consists of the amino acid sequence shown in SEQ ID NO.2, and the sequence of the CDR3 region consists of the amino acid sequence shown in SEQ ID NO. 3.

2. The nanobody of claim 1, wherein the variable region sequence of the nanobody consists of the amino acid sequence set forth in SEQ id No. 4.

3. A fusion protein of the nanobody of claim 2 and human placental alkaline phosphatase, wherein the fusion protein is formed by tandem connection of the nanobody and human placental alkaline phosphatase.

4. A polynucleotide molecule encoding the nanobody of claim 2, wherein the sequence of the polynucleotide molecule is represented by SEQ ID No. 5.

5. An expression vector comprising the polynucleotide molecule of claim 4, wherein said vector is pMES 4.

6. A host cell comprising the expression vector of claim 5, wherein said cell is E.coli BL21(DE 3).

7. Use of the nanobody of claim 1 or 2 for the preparation of a procalcitonin detection kit.

8. The procalcitonin detection method based on the non-diagnosis purpose is characterized in that the method is a double-antibody sandwich enzyme-linked immunoassay, the variable region sequence of the first antibody is shown as SEQ ID No.6 or SEQ ID No.9, the second antibody is an enzyme-linked second antibody, and the variable region sequence of the second antibody is shown as the amino acid sequence of SEQ ID No. 4.

9. The method of claim 8, wherein the enzyme-linked secondary antibody is a fusion protein of a nanobody against procalcitonin and alkaline phosphatase.

10. An immunoassay kit for detecting procalcitonin by using a double-antibody sandwich method, the kit comprises a first antibody for capturing an antigen and a second antibody for binding with the antigen to trigger an enzyme-linked reaction, and is characterized in that the variable region sequence of the first antibody is shown as SEQ ID No.6 or SEQ ID No.9, the second antibody is a fusion protein of a nano antibody for resisting procalcitonin and alkaline phosphatase, and the sequence of the fusion protein is shown as the amino acid sequence of SEQ ID No. 8.

Technical Field

The invention discloses a nano antibody, and belongs to the technical field of polypeptides.

Background

Procalcitonin (PCT) is a hormone-inactive calcitonin propeptide material consisting of a calcitonin, an N-terminal residue fragment and having a relative molecular weight of 13 KDa. Calcitonin is only produced when thyroid C cells are stimulated by hormones, whereas PCT can be secreted by different cell types of many organs after being stimulated by pro-inflammatory responses, particularly by bacteria.

PCT can be an important marker that specifically distinguishes between bacterial infections and inflammatory responses due to other causes, and its levels in plasma are elevated when severe bacterial, fungal, parasitic infections, as well as sepsis and multi-organ failure. Clinical data show that when the concentration of PCT is more than 0.1ng/ml, clinically relevant bacterial infection exists, and antibiotics are required to be used for treatment; when PCT concentrations are greater than 0.5ng/ml, the risk that the patient may develop severe sepsis or septic shock is considered. PCT reflects the activity of the systemic inflammatory response. Factors that influence PCT levels include the size and type of the organ being infected, the type of bacteria, the degree of inflammation and the status of the immune response. The change of the PCT concentration in a case is detected, thereby being beneficial to judging the bacterial infection condition of an organism and judging the health condition of the organism and having important significance for clinical and basic researches.

Since the last 80 s, the immunological detection technology has rapidly developed with the maturity of monoclonal antibodies, artificially synthesized polypeptides, genetically engineered expressed antigens and various labeling technologies, and conventional immunoprecipitation and immunoagglutination are gradually replaced by immunoturbidimetry, rate-scattering turbidimetry, latex-enhanced transmission turbidimetry and chemiluminescence analysis technology, so that the detection is faster. At present, there are many methods for detecting PCT, and the PCT can be not only qualitative but also quantitative. The following methods are commonly used:

1. radioimmunoassay: the method utilizes the polyclonal antibody which is artificially synthesized to specifically recognize and connect to synthesize the amino acid procalcitonin. The method can detect the serum PCT of normal people with the sensitivity of 4pm/ml, and detect the mixture of free PCT, bound PCT and calcitonin gene-related peptide precursor, but can not distinguish the three substances. And the detection of the method takes 19-22h and has the risk of radioactive element pollution.

2. A colloidal gold colorimetric method: the method utilizes colloidal gold labeled anti-PCT monoclonal antibodies and anti-PCT polyclonal antibodies for coating. When serum or plasma is added to the sample wells, the gold-labeled monoclonal antibodies bind to PCT in the sample, forming gold-labeled antigen-antibody complexes. The complex migrates on the reaction membrane and binds to the anti-PCT antibody immobilized on the membrane to form a larger complex. When the concentration of PCT exceeds 0.5ng/ml, the composite shows red, the shade of red is proportional to the concentration of PCT, and the concentration range of PCT can be obtained by comparing with a standard colorimetric plate. However, this method is prone to large errors in the color contrast process.

3. Transmission immunoturbidimetry: the principle of the method is that PCT in a sample and PCT monoclonal antibody in a reagent generate antigen-antibody reaction to increase the turbidity of reaction liquid, the turbidity of the reaction liquid and the amount of the added antigen are in a linear relation in a certain range, and a biochemical analyzer or other optical detection instruments are used for measuring the absorbance value of the reaction liquid at the 600nm wavelength. The absorbance value measured is directly proportional to the concentration of PCT detected. Although the method is simple and rapid, the methodology and clinical application of immunoturbidimetry still need further verification.

4. Double antibody sandwich immunochemiluminescence: the method employs a double monoclonal antibody, one of which is a calcitonin antibody and the other of which is an anti-calcin antibody, which bind to the calcitonin and anti-calcin sites of the PCT molecule, respectively, to preclude cross-reactivity. One of the antibodies is light-labeled, the other is unlabeled and fixed on the inner wall of the reaction vessel, the two antibodies are combined with PCT molecules to form a sandwich complex in the reaction process, and the light-emitting part is positioned on the surface of the reaction vessel. The method has the advantages of simple operation, strong specificity and high sensitivity, the measured bottom limit value can reach 0.1ng/ml, and the time is taken for 2 hours.

Based on the outstanding characteristics of PCT in clinical diagnosis, the development of specific binding antibodies against PCT, and increasing the detection range while ensuring sensitivity, is an urgent need in the art.

In 1993, Hamers-Casterman et al found that a class of heavy chain-only dimers (H) was found in camelids (camels, dromedary and llamas) in vivo₂) Antibodies of the type IgG2 and IgG3, which are predominantly of the IgG2 and IgG3, are also referred to as single domain antibodies or single domain antibodies (sdabs) because they lack a light chain and are thus referred to as Heavy chain-only antibodies (HCAbs), whereas their antigen binding site consists of one domain, referred to as a VHH region. Since this type of antibody is a variable region sequence after removal of a constant region, the molecular weight is only 15kD, and the diameter is about 10 nm, and thus it is also called nanobody (Nbs). In addition, such single domain antibodies, called VNARs, are also observed in sharks. This heavy chain-only antibody was originally recognized only as a pathological form of a human B-cell proliferative disease (heavy chain disease). This heavy chain-only antibody may be due to genomic level mutations and deletions that result in the inability of the heavy chain CH1 domain to be expressed, such that the expressed heavy chain lacks CH1 and thus lacks the ability to bind to the light chain, thus forming a heavy chain dimer.

Nanobodies are comparable in affinity to their corresponding scFv, but surpass scfvs in solubility, stability, resistance to aggregation, refolding, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination, relative to scfvs of conventional four-chain antibodies.

Nanobodies have minimal functional antigen-binding fragments derived from HCabs in adult camelids, have high stability and high avidity for antigen binding, and can interact with protein clefts and enzymatic active sites, making their action similar to inhibitors. Therefore, the nano-antibody can provide a new idea for designing small molecule enzyme inhibitors from peptide-mimetic drugs. Due to the heavy chain only, nanobodies are easier to manufacture than monoclonal antibodies. The unique properties of nanobodies, such as stability in extreme temperature and pH environments, allow for large yields to be produced at low cost. Therefore, the nano antibody has great value and development prospect in the treatment and diagnosis of diseases.

In view of that PCT is more over-expressed in some serious bacterial, fungal and parasitic infections, sepsis, multi-organ failure and other diseases, the development of the nano antibody for resisting PCT fully exerts the super-strong antigen recognition capability of the nano antibody, and particularly recognizes some antigenic determinants hidden in fissures or cavities to form a new requirement in the technical field of antibodies. However, the existence of some structural and functional defects such as low affinity, easy aggregation, short serum half-life, etc. due to the low molecular weight of the nanobody prevents the further application of the nanobody. In the specific application of PCT immunoassay, if the anti-PCT antibody recognizes PCT epitope singly or with sites close to or overlapping, the specific antigen-antibody binding reaction is affected, thereby seriously affecting the detection efficiency. The invention aims to provide an anti-PCT nano antibody which can fully exert the excellent performance of the nano antibody and overcome the inherent defects of the nano antibody, namely, the antibody has a unique epitope recognition site, can recognize and combine antigens with high specificity and can obtain excellent detection efficiency in the immunoassay of the PCT antigens, particularly in a double-antibody sandwich method.

Disclosure of Invention

Based on the above objects, the present invention provides a procalcitonin-resistant nanobody, wherein the variable region of the nanobody has 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the sequence of CDR1 region consists of the amino acid sequence shown in SEQ ID No.1, the sequence of CDR2 region consists of the amino acid sequence shown in SEQ ID No.2, and the sequence of CDR3 region consists of the amino acid sequence shown in SEQ ID No. 3. The antibodies have unique epitope recognition sites.

In a preferred technical scheme, the variable region sequence of the nanobody consists of the amino acid sequence shown in SEQ ID NO. 4. One preferred example of a nanobody having this variable region sequence in the present invention is nanobody 6H 6.

Secondly, the invention provides the fusion protein of the nano antibody and the human placenta alkaline phosphatase, and the fusion protein is formed by connecting the nano antibody and the human placenta alkaline phosphatase in series.

Thirdly, the invention also provides a polynucleotide molecule for coding the nano antibody, and the sequence of the polynucleotide molecule is shown by SEQ ID NO. 5.

Fourthly, the invention provides an expression vector containing the polynucleotide molecule, wherein the vector is pMES 4.

Fifth, the present invention provides a host cell comprising the above expression vector, said cell being E.coli BL21(DE 3).

Sixth, the invention also provides application of the nano antibody in preparation of a procalcitonin detection kit.

Seventhly, the invention provides a procalcitonin detection method based on non-diagnostic purposes, which is a double-antibody sandwich enzyme-linked immunoassay, wherein the variable region sequence of the first antibody is shown as SEQ ID NO.6 or SEQ ID NO.9, the second antibody is an enzyme-linked second antibody, and the variable region sequence of the second antibody is shown as the amino acid sequence of SEQ ID NO. 4.

In a preferred embodiment, the enzyme-linked second antibody is a fusion protein of a procalcitonin-resistant nanobody and alkaline phosphatase.

Finally, the invention also provides an immunoassay kit for detecting PCT by using a double-antibody sandwich method, which comprises a first antibody for capturing an antigen and a second antibody for binding with the antigen to trigger an enzyme-linked reaction, and is characterized in that the variable region sequence of the first antibody is shown as SEQ ID No.6 or SEQ ID No.9, the second antibody is a fusion protein of a nano antibody for resisting procalcitonin and alkaline phosphatase, and the sequence of the fusion protein is shown as the amino acid sequence of SEQ ID No. 8.

The anti-PCT nano antibody 6H6 provided by the invention has a unique epitope recognition site, has specific recognition and binding capacity on PCT antigen, has higher antigen affinity which can reach 3.773E-9, recognizes different epitopes, can be combined with other nano antibodies to be applied to a double-antibody sandwich enzyme-linked immunoassay method for detecting procalcitonin, and in a preferred embodiment, the nano antibody 6H6 is respectively combined with two strains of nano antibodies BF5 and 2H4 to be used, so that an excellent detection effect is shown.

Drawings

FIG. 1 shows the electrophoretic identification of total RNA extracted;

FIG. 2 shows the first round of PCR amplification of antibody variable region gene electrophoresis identification map;

FIG. 3 is the second round of PCR amplification of antibody variable region gene electrophoresis identification map;

FIG. 4 is a schematic diagram of the structure of the pMES4 expression vector;

FIG. 5 shows the electrophoretic identification chart of the product of the double digestion reaction with pMES4 vector;

FIG. 6 shows the electrophoretic identification chart of the transformant identified by colony PCR;

FIG. 7 is a SDS-PAGE pattern of nanobody purification;

FIG. 8 is a flow chart of Biacore analysis of nanobody binding sites.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.