CN-121975755-A - Lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody, preparation method and application

CN121975755ACN 121975755 ACN121975755 ACN 121975755ACN-121975755-A

Abstract

The invention relates to the field of biological medicine, in particular to a lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody, a preparation method and application thereof, wherein based on the lactic dehydrogenase LDHB protein 196 serine phosphorylating polypeptide, the lactic dehydrogenase LDHB protein 196 serine phosphorylating antigen and antibody are prepared, the preparation method of the lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody is provided, the prepared lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody can detect low-abundance LDHB Ser 196 signals, and the lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody is suitable for various detection platforms such as Western blot and immunohistochemistry, and meets the detection requirements of tumor tissue samples.

Inventors

CHENG XIAWEI
SHANG MENGRAN
FENG JIAN
SUN WEIXIA
Ying Lingxuan
ZHU YIFEI
WANG LUQING
YANG WENLAN

Assignees

华东理工大学

Dates

Publication Date: 20260505
Application Date: 20260209

Claims (10)

1. A serine phosphorylating polypeptide of lactic dehydrogenase LDHB protein 196 is characterized in that the amino acid sequence of the polypeptide is LGEHGDS (pi) SVAVWSGV.
2. A serine-phosphorylated antigen at position 196 of a lactate dehydrogenase LDHB protein, characterized in that the antigen is formed by crosslinking a serine-phosphorylated polypeptide at position 196 of the lactate dehydrogenase LDHB protein with KLH or BSA according to claim 1.
3. A serine phosphorylating antibody at position 196 of lactic dehydrogenase LDHB protein, which is prepared by emulsifying a serine phosphorylating antigen at position 196 of lactic dehydrogenase LDHB protein according to claim 2 and immunizing in animals.
4. A preparation method of a lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody is characterized by comprising the following steps of Preparation of serine-phosphorylated antigen at position 196 of LDHB protein the serine-phosphorylated polypeptide at position 196 of LDHB protein according to claim 1 is crosslinked to KLH and/or BSA to obtain antigen: step 2, animal immunization, namely immunizing rabbits after antigen emulsification to obtain immunized rabbit serum; Step 3, determining antibody titer of the antibody in the serum of the immunized rabbit; And 4, purifying the antibody to obtain the lactic dehydrogenase LDHB protein 196 serine phosphorylated antibody.
5. The method for preparing the lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody according to claim 4, wherein the method for emulsifying the antigen in the step 2 animal immunization is to take the LDHB protein 196 serine phosphorylating antigen according to claim 2, and fully mix and emulsify the same with an equal volume of adjuvant, wherein the first immunization uses a complete adjuvant, and the subsequent immunization uses an incomplete adjuvant.
6. The method for producing serine phosphorylating antibody at position 196 of lactate dehydrogenase LDHB protein according to claim 4, wherein the antibody titer in step 3 is measured by ELISA indirect detection method.
7. The method for producing a serine phosphorylating antibody at position 196 of lactate dehydrogenase LDHB protein according to claim 4, wherein the step 4 antibody purification step comprises: (1) protein A affinity purification, namely filtering immunized rabbit serum, loading the filtered rabbit serum to an equilibrated protein A affinity chromatographic column, and eluting with eluent after full adsorption to collect elution components to obtain crude extracted IgG; (2) Loading the crude extracted IgG to a phosphorylation affinity chromatographic column for forward screening, and collecting eluting components to obtain a target antibody; (3) And (3) carrying out non-phosphorylated polypeptide affinity purification, namely loading the target antibody obtained in the step (2) to a non-phosphorylated affinity chromatographic column for negative screening, and collecting effluent liquid to obtain the target antibody with high specificity.
8. The method for producing a serine phosphorylating antibody at position 196 of lactate dehydrogenase LDHB protein according to claim 4, wherein the step 4 antibody purification step comprises: (1) Filtering immune rabbit serum with a filter membrane, loading the filtered immune rabbit serum onto a protein A affinity chromatographic column which is balanced after washing, eluting the protein A affinity chromatographic column by using eluent to remove non-IgG impurity proteins, eluting the protein A affinity chromatographic column by using acidic eluent, collecting eluent, and immediately regulating the pH value of an elution component to be neutral by using a neutralization buffer solution to obtain an elution product of protein A purification, namely crude extracted IgG; (2) Washing the phosphorylated polypeptide affinity chromatographic column, loading the crude extracted IgG obtained in the step (1) to the phosphorylated polypeptide affinity chromatographic column, eluting to remove unbound and nonspecific binding impurity proteins, eluting the phosphorylated polypeptide affinity chromatographic column by using a low pH eluting buffer solution, and immediately adding a neutralizing buffer solution to adjust the pH of the collected eluent to be neutral to obtain a solution rich in phosphorylated site specific antibodies, namely a target antibody solution; (3) And (3) affinity purification of the non-phosphorylated polypeptide, namely cleaning a non-phosphorylated polypeptide affinity chromatographic column, loading the antibody obtained by purification in the step (2) to an affinity chromatographic column taking the non-phosphorylated antigen polypeptide as a ligand, collecting a flow-through component, and regulating the pH value to 7.0 to obtain the high-specificity phosphorylated antibody.
9. Use of the lactate dehydrogenase LDHB protein 196 serine-phosphorylated polypeptide of claim 1, the lactate dehydrogenase LDHB protein 196 serine-phosphorylated antigen of claim 2, and the lactate dehydrogenase LDHB protein 196 serine-phosphorylated antibody of claim 3 for the preparation of a tumor metabolism detection reagent.
10. A kit comprising the serine phosphorylating antibody at position 196 of lactic dehydrogenase LDHB protein according to claim 3, and/or a tumor metabolism detecting reagent comprising the serine phosphorylating antibody at position 196 of lactic dehydrogenase LDHB protein according to claim 9.

Description

Lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody, preparation method and application Technical Field The invention relates to the field of biological medicine, in particular to a lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody, a preparation method and application thereof. Background Cell metabolic reprogramming is one of the core features of tumors, where the warburg effect is particularly pronounced-tumor cells preferentially produce large amounts of lactic acid via the glycolytic pathway even under oxygen-deficient conditions. Lactate dehydrogenase B (LDHB) is a key rate-limiting enzyme catalyzing this metabolic process, exhibits significantly high expression in a variety of malignancies, whose active state directly determines lactate production efficiency and is closely related to tumor progression, metastasis and poor prognosis in patients. Studies have shown that the phosphorylation modification of serine 196 (S196) of LDHB is a key molecular switch that regulates its enzymatic activity. The specific phosphorylation of the site can obviously enhance the catalytic efficiency of LDHB by inducing the spatial conformational change of the LDHB, thereby directly promoting lactic acid generation. In the tumor microenvironment, the phosphorylation level of the LDHB S196 locus is obviously positively correlated with glycolytic flux, and becomes an important molecular marker reflecting the metabolic state of tumor cells, and the phosphorylation modification plays a key regulation role in physiological conditions such as exercise stress and the like, so that the phosphorylation modification has general biological significance in metabolic steady state maintenance. However, the lack of a high-affinity antibody tool capable of specifically recognizing the LDHB S196 phosphorylation modification at present severely restricts the deep analysis of the biological function of the modification site and the clinical transformation application thereof. The existing detection means is difficult to realize accurate identification and quantitative analysis of the key phosphorylation event, so that the specific action mechanism of LDHB S196 phosphorylation in tumor metabolism regulation is not yet elucidated, and the potential value of the LDHB S196 serving as a tumor diagnosis marker and a treatment target point cannot be fully estimated. Along with the deep research on the phosphorylation function of LDHB S196, the application value of the modified site in the fields of early diagnosis of tumor, dynamic evaluation of metabolic state, targeted intervention and the like is increasingly prominent. However, there is no high affinity antibody tool capable of specifically recognizing the phosphorylation modification of LDHB S196, which severely restricts the intensive development of related studies. Therefore, there is a need to develop a specific detection tool for the key modification site, and the development of this detection tool not only helps to elucidate the molecular regulatory mechanism of tumor metabolic reprogramming, but also provides important technical support for exploring novel therapeutic strategies based on metabolic intervention. Disclosure of Invention The invention aims to provide a serine phosphorylating antibody at 196 th serine of a lactate dehydrogenase LDHB protein. The invention also aims to provide a preparation method and application of the lactic dehydrogenase LDHB protein 196 serine phosphorylating antibody. On one hand, the lactic dehydrogenase LDHB protein 196 serine phosphorylation antibody provides key technical support for systematically researching the regulation mechanism of LDHB phosphorylation modification and analyzing the molecular function of the antibody in the tumor metabolic process, on the other hand, the accurate evaluation of the LDHB S196 phosphorylation level of a tumor patient can be realized based on the antibody, a novel molecular marker is provided for preparing detection reagents for early tumor diagnosis, metabolic typing, prognosis judgment and the like, and meanwhile, the successful development of the antibody lays an important foundation for screening small molecular inhibitors for targeting LDHB S196 phosphorylation modification and exploring a novel strategy for tumor metabolism targeting treatment. The technical scheme adopted for realizing the purpose of the invention is as follows: in a first aspect, the application relates to a serine phosphorylating polypeptide at position 196 of a lactate dehydrogenase LDHB protein, said polypeptide being phosphorylated at position 196, said polypeptide having the amino acid sequence LGEHGDS (pi) SVAVWSGV. In a second aspect, the application relates to a serine phosphorylating antigen at position 196 of a lactate dehydrogenase LDHB protein, said antigen being formed by cross-linking a serine phosphorylating polypeptide at position 196 of said lactate dehydrogenase LDHB protein with KLH o