阅读说明：本技术 一种人AD7c-NTP单克隆抗体及其制备方法和应用 (Human AD7c-NTP monoclonal antibody and preparation method and application thereof ) 是由 赵风强 董兵 杨春 吴妤 邬晓东 于 2020-02-26 设计创作，主要内容包括：本发明公开了一种人AD7c-NTP单克隆抗体及其制备方法和应用,通过以下步骤制备：以AD7c-NTP重组蛋白为免疫原,免疫BALB/c小鼠,再进行小鼠脾细胞和鼠骨髓瘤细胞融合及亚克隆,筛选特异性、效价高的单克隆抗体,最后腹水制备及纯化,获得抗人AD7c-NTP蛋白的单克隆抗体。本发明与现有的技术相比的优点在于：本发明提供了可产生抗人AD7c-NTP单克隆抗体的杂交瘤细胞株I1B10和杂交瘤细胞株X1C11,以及抗人AD7c-NTP单克隆抗体I1B10和X1C11,该抗体对可通过双抗体夹心法对人体液中AD7c-NTP浓度进行检测,具有重要的应用前景。(The invention discloses a human AD7C-NTP monoclonal antibody and a preparation method and application thereof, and the monoclonal antibody is prepared by the following steps of immunizing a BA L B/C mouse by taking AD7C-NTP recombinant protein as immunogen, then carrying out fusion and subcloning of mouse spleen cells and mouse myeloma cells, screening the monoclonal antibody with high specificity and potency, and finally preparing and purifying ascites to obtain the monoclonal antibody resisting human AD7C-NTP protein.)

1. A process for preparing the monoclonal antibody of anti-human AD7c-NTP protein includes such steps as immunizing BA L B/c mouse with recombinant AD7c-NTP protein as immunogen, fusing and subcloning the splenocyte and myeloma cell of mouse, screening the monoclonal antibody with high specificity and potency, preparing ascites and purifying to obtain the monoclonal antibody of anti-human AD7c-NTP protein.

2. The method of claim 1, wherein the monoclonal antibody against human AD7c-NTP is prepared by the following steps: the AD7c-NTP recombinant protein has a sequence of SEQ ID NO.1, and the preparation method is as follows: human AD7c-NTP gene (SEQ ID NO.2) is inserted into an expression vector pET30a, pET30a-AD7c-NTP fusion expression plasmid is constructed, escherichia coli is transformed, and then IPTG is used for induction, purification and renaturation to obtain the human AD7c-NTP recombinant protein.

3. The method for preparing a monoclonal antibody against human AD7c-NTP according to claim 1, wherein the BA L B/c mouse is a female mouse aged 8-12 weeks.

4. The method for preparing the monoclonal antibody against human AD7c-NTP according to claim 1, wherein the monoclonal antibody against human AD7c-NTP is obtained by high throughput screening using E L ISA method (enzyme linked immunosorbent assay).

5. The method of claim 1, wherein the monoclonal antibody against human AD7c-NTP is prepared by the following steps: the purification method is Protein A affinity purification.

6. A monoclonal antibody against human AD7c-NTP protein, characterized by: the monoclonal antibody against human AD7c-NTP protein is prepared by the preparation method according to any one of claims 1-5.

7. A method for screening an AD7c-NTP antibody pair, which is characterized by comprising the following steps: and (3) carrying out AD7c-NTP antigen detection by adopting a chessboard titration method, and selecting a pairing antibody with better sensitivity and specificity.

8. A method for detecting the concentration of AD7c-NTP is characterized in that: the concentration of AD7C-NTP in human tissues, cells or body fluids can be detected by a mouse anti-human AD7C-NTP monoclonal antibody I1B10 and a mouse anti-human AD7C-NTP monoclonal antibody X1C11 based on an immunization method, and preferably, the concentration of human AD7C-NTP in urine samples is detected.

9. The method according to claim 8, wherein the concentration of AD7c-NTP is determined by: the body fluid includes urine, cerebrospinal fluid, serum or blood.

Technical Field

The invention relates to the field of monoclonal antibody preparation, in particular to a human AD7c-NTP monoclonal antibody and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is a neurodegenerative disease characterized by a chronic worsening process of impaired mental function and memory loss. In 2001, 1100 million people worldwide had alzheimer's disease (Gadit, 2001). By 2005, 2420 million people worldwide had senile dementia [ Reitz C,2011 ]; in 2010, The number is increased to 3600 ten thousand, in 2030 to 6600 ten thousand, in 2050 to 1.15-1.35 hundred million [ The Global Impactor Dementia 2013-. Currently, alzheimer's disease becomes the sixth leading cause of death (AA, 2012). Alzheimer's disease accounts for about 70% of all dementia patients. Recent diagnostic guidelines divide alzheimer's disease into three stages: a dementia stage; symptomatic, pre-dementia (also known as mild cognitive impairment) and asymptomatic, preclinical stages [ Jack CR, 2011 ]. There is currently no effective treatment to prevent, arrest or reverse alzheimer's disease; the only effective treatment for AD is to delay or alleviate symptoms [ Huang Y,2012 ]. Therefore, early diagnosis of AD is crucial.

Neuronal filamentous proteins (NTPs) are a class of transmembrane phosphorylated proteins that induce Neuronal apoptosis and mitochondrial dysfunction [ De la Monte SM,2001 ]; NTPs are expressed increasingly during neuronal cell proliferation, differentiation, brain development and AD neuronal degeneration [ De la Monte SM,1997 ].

AD7c-NTP is a member of NTP family, is a new cDNA containing Alu sequence, which is first separated from temporal lobe of AD late stage patient by Suzanne M.de la Monte, etc., and is named as AD7c-NTP, its sequence total length is 1442bp, contains ORF composed of 1125 nucleotides, can code mature protein containing 375 amino acids, molecular mass is about 41kD, isoelectric point is 9.89[ Monet SM, et al 1997 ]. 2004, Guijunhao et al found that the AD7c-NTP gene was located on the negative strand of lp36.11 by bioinformatics method, and there were no intron sequences, and 3 transmembrane regions of the encoded product were possible.

Ghanbari H and the like adopt an immunological method to detect the content of AD7c-NTP protein in cerebrospinal fluid (CSF) or urine of suspected AD patients, thereby being beneficial to early diagnosis and differential diagnosis of AD. De la Monte SM et al found that abnormal expression of the AD7c-NTP gene was closely related to the typical cell phenotype of AD-type dementia. AD7c-NTP may be a new biomarker for early diagnosis of AD with important clinical application value [ De la Monte SM,2002& Neugroschl J,2002 ]. Therefore, the development of the AD7c-NTP antigen detection kit for clinical diagnosis is of great significance.

In 2007, Shinesui and the like prepare a polyclonal antibody by designing and synthesizing an AD7c-NTP antigenic determinant polypeptide fragment for developing an AD7c-NTP in-vitro diagnosis kit; poplar Yongfang (2015) and the like are used for designing and synthesizing AD7c-NTP epitope polypeptide, preparing a mouse monoclonal antibody and developing an AD7c-NTP in-vitro diagnosis kit. However, the AD7c-NTP in-vitro diagnosis kit on the market has high feedback rate in clinical application, low coincidence rate with clinical symptoms, inconvenient clinical operation and long reaction time. Therefore, the development of an AD7c-NTP in-vitro diagnostic kit which has better specificity, higher clinical coincidence rate and convenient and quick operation is urgently needed.

The most central component in the AD7c-NTP in-vitro diagnosis kit is anti-AD 7c-NTP antibody raw material. Because the content of AD7c-NTP in cerebrospinal fluid and urine of human body is very small, it is difficult to obtain natural protein. Most of domestic AD7c-NTP in-vitro diagnostic kits are developed based on AD7c-NTP polypeptide antigens, and the sensitivity and specificity of the kits are insufficient, so that the acquisition of high-affinity and high-specificity anti-AD 7c-NTP antibodies is the key for successfully developing AD7c-NTP in-vitro diagnostic kits. AD7c-NTP is a membrane protein, the structure is relatively complex, at present, a large amount of AD7c-NTP protein antigens are not reported to be obtained through a DNA recombination technology, an anti-AD 7c-NTP monoclonal antibody is prepared, and an AD7c-NTP in-vitro diagnosis kit is developed.

Therefore, the invention utilizes DNA recombination technology to synthesize AD7c-NTP full-length gene, construct to prokaryotic expression vector, and obtain a large amount of AD7c-NTP protein in vitro through optimized fermentation, purification and renaturation processes; and successfully develops a monoclonal antibody of high affinity and high specificity against AD7 c-NTP; based on the method, a convenient and rapid AD7c-NTP protein diagnosis method and a diagnosis reagent thereof are developed, and the content of AD7c-NTP in cerebrospinal fluid, urine and serum can be used as early screening of AD patients so as to achieve early diagnosis of the AD patients.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the technical defects and provide an antibody specifically bound with human AD7c-NTP protein, wherein the antibody is a monoclonal antibody.

In order to solve the problems, the technical scheme of the invention is that the preparation method of the monoclonal antibody of anti-human AD7c-NTP protein comprises the following steps of immunizing a BA L B/c mouse by taking AD7c-NTP recombinant protein as immunogen, then carrying out fusion and subcloning on mouse spleen cells and mouse myeloma cells, screening the monoclonal antibody with high specificity and potency, and finally preparing and purifying ascites to obtain the monoclonal antibody of anti-human AD7c-NTP protein.

As an improvement, the AD7c-NTP recombinant protein has a sequence shown in SEQ ID NO.1, and the preparation method is as follows: human AD7c-NTP gene (SEQ ID NO.2) is inserted into an expression vector pET30a, pET30a-AD7c-NTP fusion expression plasmid is constructed, escherichia coli is transformed, and then IPTG is used for induction, purification and renaturation to obtain the human AD7c-NTP recombinant protein.

As an improvement, the BA L B/c mice were female mice 8-12 weeks old.

As an improvement, the anti-human AD7c-NTP monoclonal antibody is obtained by adopting an E L ISA method (enzyme-linked immunosorbent assay) high-throughput screening.

As an improvement, the purification method is Protein A affinity purification.

The monoclonal antibody of anti-human AD7c-NTP protein is prepared by the preparation method.

A screening method of AD7c-NTP antibody pairs adopts a chessboard titration method to carry out AD7c-NTP antigen detection, and selects a paired antibody with good sensitivity and specificity.

A method for detecting the concentration of AD7C-NTP features that the mouse-anti-human AD7C-NTP monoclonal antibody I1B10 and mouse-anti-human AD7C-NTP monoclonal antibody X1C11 are used to detect the concentration of AD7C-NTP in human tissue, cell or body fluid, preferably the concentration of human AD7C-NTP in urine sample.

As a refinement, the bodily fluid includes urine, cerebrospinal fluid, serum or blood.

Compared with the prior art, the invention has the advantages that:

provides a hybridoma cell strain I1B10 and a hybridoma cell strain X1C11 which can produce an anti-human AD7C-NTP monoclonal antibody, and anti-human AD7C-NTP monoclonal antibodies I1B10 and X1C11, the antibodies can detect the concentration of AD7C-NTP in human body fluid by a double-antibody sandwich method, and have important application prospects.

The invention provides an expression and purification method of human AD7C-NTP in vitro recombinant protein, which solves the problems that human AD7C-NTP protein is not easy to obtain in vitro and is unstable, moreover, a wider spectrum anti-AD 7C-NTP antibody can be prepared by immunizing animals with the human AD7C-NTP recombinant protein, particularly a mouse anti-human AD7C-NTP monoclonal antibody with better affinity and specificity can be prepared, in addition, the anti-human AD7C-NTP monoclonal antibody I1B10 and X1C11 are adopted, a method for detecting E L ISA by the human AD7C-NTP protein is established, high sensitivity (0.3ng/ml) and specificity of detection are realized, finally, the content of the human AD7C-NTP protein in urine can be rapidly and accurately determined, and clinical bed reference value is provided for reliable early diagnosis and early treatment of AD patients.

Drawings

FIG. 1 is a SDS-PAGE result of recombinant plasmid pET30a-AD7c-NTP small sample induced expression.

FIG. 2 is a diagram showing the results of SDS-PAGE identification after purification and renaturation of human AD7c-NTP recombinant protein.

FIG. 3 is a graph showing the linearity and sensitivity of the ISA detection method for human AD7c-NTP antigen E L.

Detailed Description

The present invention is further described below by way of specific examples, but the present invention is not limited to only the following examples. Variations, combinations, or substitutions of the invention, which are within the scope of the invention or the spirit, scope of the invention, will be apparent to those of skill in the art and are within the scope of the invention.

A process for preparing the monoclonal antibody of anti-human AD7c-NTP protein includes such steps as immunizing BA L B/c mouse with recombinant AD7c-NTP protein as immunogen, fusing and subcloning the splenocyte and myeloma cell of mouse, screening the monoclonal antibody with high specificity and potency, preparing ascites and purifying to obtain the monoclonal antibody of anti-human AD7c-NTP protein.

The AD7c-NTP recombinant Protein has a sequence of SEQ ID NO.1, and the preparation method comprises the steps of inserting a human AD7c-NTP gene (SEQ ID NO.2) into an expression vector pET30a, constructing pET30a-AD7c-NTP fusion expression plasmid, transforming escherichia coli, inducing by IPTG, purifying and renaturing to obtain the human AD7c-NTP recombinant Protein, wherein a BA L B/c mouse is a female mouse with the age of 8-12 weeks, and an anti-human AD7c-NTP monoclonal antibody is obtained by adopting an E L ISA method (enzyme-linked immunosorbent assay) high-throughput screening, and the purification method is Protein A affinity purification.

The monoclonal antibody of anti-human AD7c-NTP protein is prepared by the preparation method.

A screening method of AD7c-NTP antibody pairs adopts a chessboard titration method to carry out AD7c-NTP antigen detection, and selects a paired antibody with good sensitivity and specificity.

A method for detecting the concentration of AD7C-NTP features that the mouse-anti-human AD7C-NTP monoclonal antibody I1B10 and mouse-anti-human AD7C-NTP monoclonal antibody X1C11 are used to detect the concentration of AD7C-NTP in human tissue, cell or body fluid, preferably human AD7C-NTP in urine specimen.