CN-122011137-A - Antigen polypeptide for preparing specificity binding HERVH capsid protein, polyclonal antibody and application thereof

Abstract

The invention provides an antigen polypeptide and polyclonal antibody for preparing specificity binding HERVH capsid protein and application thereof, wherein the amino acid sequence of the antigen polypeptide is shown as SEQ ID NO. 2. The high-titer specific antibody is obtained by preparing the polypeptide into an immunogen for immunizing animals and purifying. The antibody can specifically identify HERVH capsid proteins in tissue and body fluid samples (such as serum and exosomes). The antibody and the kit provided by the invention have high sensitivity and strong specificity, can be used for early diagnosis, auxiliary diagnosis, prognosis evaluation and risk stratification of colorectal cancer, and have remarkable clinical application value.

Inventors

• SHEN JIANFENG
• LI QIAN
• SONG PING
• Zheng Qiangting

Assignees

• 上海交通大学医学院附属第九人民医院

Dates

Publication Date

20260512

Application Date

20260213

Claims (10)

1. 1. An antigen polypeptide for preparing a polyclonal antibody specifically binding to HERVH capsid protein, characterized in that the amino acid sequence of the antigen polypeptide is shown in SEQ ID NO. 2.
2. 2. An immunogen for preparing polyclonal antibodies which specifically bind to HERVH capsid proteins, characterized in that it is prepared by using the antigen polypeptide of claim 1 as hapten.
3. 3. The immunogen according to claim 2, wherein the preparation method is characterized in that the antigen polypeptide according to claim 1 is mixed with a carrier protein which is maleylated in advance in a buffer solution with the pH of 6.5-7.5, wherein the molar ratio of the antigen polypeptide to the carrier protein is 20:1-50:1, the mixture is reacted for 1-3 hours at room temperature, and unconjugated antigen polypeptide is removed by dialysis or gel filtration after the reaction is finished, so that the immunogen is obtained.
4. 4. Polyclonal antibodies for specific detection of HERVH capsid proteins, characterized in that they are obtained by immunization of a non-human mammal with an immunogen according to claim 2 or 3.
5. 5. The method for producing a polyclonal antibody according to claim 4, comprising the steps of: S1, immunizing a non-human mammal with the immunogen of claim 2 or 3, and detecting and screening high-titer immunized animals; s2, collecting whole blood of the selected immunized animal, separating serum, and carrying out antigen affinity purification by utilizing a solid phase medium coupled with the polypeptide shown in SEQ ID NO. 2 to obtain the polyclonal antibody.
6. 6. The preparation method of claim 5, wherein the immunized animal is an immunized rabbit, the number of times of immunization is 3-5, and the immunized animal detected and screened refers to the immunized rabbit with the antigen polypeptide detection titer reaching more than 1:10000 by adopting an ELISA method.
7. 7. Use of the polyclonal antibody of claim 4 for the preparation of a product for diagnosis, diagnosis assistance, prognosis assessment or colorectal cancer monitoring.
8. 8. The use according to claim 7, wherein the product is used for detecting the expression level of HERVH capsid proteins in a biological sample by an immunological reaction, said immunological reaction comprising ELISA, western-blot or immunohistochemistry.
9. 9. The use according to claim 8, wherein the biological sample is selected from tumor tissue, paracancestral tissue, blood, serum, plasma, exosomes or cell culture supernatants.
10. 10. A colorectal cancer diagnostic kit, comprising the polyclonal antibody of claim 4.

Description

Antigen polypeptide for preparing specificity binding HERVH capsid protein, polyclonal antibody and application thereof Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to an antigen polypeptide and a polyclonal antibody for preparing a specific binding HERVH capsid protein and application thereof. Background Colorectal cancer (Colorectal Cancer, CRC) is a worldwide high-grade malignancy with both morbidity and mortality leading. Epidemiological data indicate that early diagnosis of colorectal cancer is critical to improving patient survival. Currently, clinical diagnosis of colorectal cancer relies mainly on colonoscopy, pathology biopsy and detection of serum tumor markers (e.g. CEA, CA 19-9). However, colonoscopy has the defects of invasiveness, high cost, poor patient compliance and the like, and is difficult to be used as a first choice means for large-scale screening, and the sensitivity and specificity of the existing serum markers in early diagnosis of CRC are often insufficient, so that missed diagnosis or misdiagnosis is easy to occur. Therefore, finding novel, highly sensitive and specific biomarkers, especially markers that can be used for non-invasive detection, is of great clinical importance. Human endogenous retroviruses (Human Endogenous Retroviruses, HERVs) account for about 8% of the human genome, and are genetic markers left behind after infection of human germ cells by ancient retroviruses. Of these, HERVH (H family endogenous retrovirus) is one of the subfamilies with higher activity. The existing studies indicate that HERVH transcripts (mRNAs) play a role in the maintenance of pluripotency in embryonic stem cells and exhibit abnormally high transcript levels in various tumor tissues such as colorectal cancer. Although the mRNA of HERVH has been reported to be up-regulated in colorectal cancer, current detection approaches have focused mainly on nucleic acid levels (e.g.qPCR detection). However, mRNA levels do not always coincide exactly with protein levels, and nucleic acid detection has certain limitations in sample handling and stability. More importantly, there is no systematic study on the expression pattern of the HERVH env gene-encoded capsid protein in colorectal cancer, whether it assembles into virus-like particles and is released extracellular, and whether it can serve as a diagnostic marker for protein levels. This is due in large part to the lack of high specificity, high affinity antibody reagents against HERVH capsid proteins. Because of the numerous HERV family members and high sequence similarity, there is great technical difficulty in preparing antibodies that specifically recognize HERVH capsid proteins without cross-reacting with other family members. The lack of effective immunological detection tools limits the development and application of HERVH proteins as clinical diagnostic markers. Disclosure of Invention The invention aims to provide an antigen polypeptide and a polyclonal antibody for preparing a specific binding HERVH capsid protein and application thereof, and provides a brand-new, sensitive and specific protein level detection means for screening, diagnosing and prognosis evaluating colorectal cancer, and has wide clinical application prospect. In a first aspect, the invention provides an antigenic polypeptide for preparing a polyclonal antibody specifically binding to HERVH capsid proteins, said antigenic polypeptide having the amino acid sequence shown in SEQ ID NO. 2. In a second aspect, the present invention provides an immunogen for preparing polyclonal antibodies specifically binding to HERVH capsid proteins, prepared using the above antigen polypeptide as hapten. In one embodiment of the invention, the preparation method of the immunogen comprises the steps of mixing the antigen polypeptide and the carrier protein which is maleylated in advance in a buffer solution with the pH of 6.5-7.5, wherein the molar ratio of the antigen polypeptide to the carrier protein is 20:1-50:1, reacting for 1-3 hours at room temperature, and removing unconjugated antigen polypeptide through dialysis or gel filtration after the reaction is finished to obtain the immunogen. In a third aspect, the invention provides a polyclonal antibody for specific detection HERVH of capsid proteins, obtained by immunization of a non-human mammal with an immunogen as described above. According to a fourth aspect of the present invention, there is provided a method for preparing the polyclonal antibody described above, comprising the steps of: s1, immunizing a non-human mammal by using the immunogen, and detecting and screening the immunized mammal with high titer; s2, collecting whole blood of the selected immunized animal, separating serum, and carrying out antigen affinity purification by utilizing a solid phase medium coupled with the polypeptide shown in SEQ ID NO. 2 to obtain the polyclonal antibody. In one embodiment