阅读说明：本技术 抗产气荚膜梭菌α毒素全人源单分子抗体7D-mFc及其应用 (Fully human monomolecular antibody 7D-mFc for resisting clostridium perfringens alpha toxin and application thereof ) 是由 张国利 田园 岳玉环 陈萍 刘楚含 吴广谋 李泽鸿 刘雨玲 王冬冬 雍伟 邓欣 于 2020-06-11 设计创作，主要内容包括：本发明公开了抗产气荚膜梭菌α毒素全人源7D-mFc单分子抗体及其应用,它的碱基序列如SEQ ID NO.3所示；氨基酸序列如SEQ ID NO.4所示；抗产气荚膜梭菌α毒素全人源7D-mFc单分子抗体在制备治疗和预防产气荚膜梭菌α毒素中毒的药物的应用；一种产气荚膜梭菌α毒素的检测试剂,它包括氨基酸序列如SEQ ID NO.4所示的产气荚膜梭菌α毒素全人源7D-mFc单分子抗体。(The invention discloses a fully human 7D-mFc monomolecular antibody for resisting clostridium perfringens alpha toxin and application thereof, wherein the base sequence of the antibody is shown as SEQ ID NO. 3; the amino acid sequence is shown as SEQ ID NO. 4; the application of the fully human 7D-mFc monomolecular antibody for resisting the clostridium perfringens alpha toxin in preparing the medicine for treating and preventing the clostridium perfringens alpha toxin poisoning; a detection reagent for clostridium perfringens alpha toxin comprises a clostridium perfringens alpha toxin fully-humanized 7D-mFc monomolecular antibody with an amino acid sequence shown as SEQ ID NO. 4.)

1. The base sequence of the fully human 7D-mFc monomolecular antibody for resisting clostridium perfringens alpha toxin is shown as SEQ ID NO. 1.

2. The amino acid sequence of the fully human 7D-mFc monomolecular antibody for resisting clostridium perfringens alpha toxin is shown as SEQ ID NO. 2.

3. Application of the fully human 7D-mFc monomolecular antibody for resisting clostridium perfringens alpha toxin in preparing a medicament for treating and preventing clostridium perfringens alpha toxin poisoning.

4. A detection kit for detecting clostridium perfringens alpha toxin comprises a clostridium perfringens alpha toxin fully human 7D-mFc monomolecular antibody with an amino acid sequence shown as SEQ ID NO. 2.

Technical Field

The invention belongs to the fields of bioengineering and disease prevention and treatment, and particularly relates to a preparation method and application of a fully human monomolecular antibody 7D-mFc for resisting clostridium perfringens alpha toxin.

Background

Clostridium perfringens (C. perfringens), also known as clostridium welchii (C. welchii), is widely found in the natural environment and found in the digestive tract of almost all warm-blooded animals, and is a member of the normal flora in the intestinal tracts of humans and animals. The clostridium perfringens can cause diseases such as lamb dysentery, necrotic enteritis and enterotoxemia of lambs, calves, piglets, rabbits and chicks, has acute morbidity and high mortality, is an important disease seriously harming the breeding industry, is a main pathogen of sudden death disease of domestic animals in recent years, and brings huge economic loss to the development of livestock industry of various countries. Various types of clostridium perfringens produce alpha toxin (CPA), the bacterium can cause human gas gangrene and enterotoxemia and necrotic enteritis of various animals, and alpha toxin antibodies are produced in livestock after the livestock are infected, so the alpha toxin antibodies are one of important bases for diagnosing clostridium perfringens diseases.

The bacterium is classified into A, B, C, D, E toxin type according to main pathogenic toxins of alpha, beta, and iota, wherein alpha toxin is one of the most important pathogenic toxins produced by each type of clostridium perfringens, has phospholipase C (PLC), sphingomyelinase (SMase) and various biological activities, and is also the most important virulence factor of clostridium perfringens type a. In recent years, research on clostridium perfringens alpha toxin mainly focuses on gene expression, enzymatic activity analysis, site-directed mutation on amino acid, action mode of alpha toxin and target cells and the like, but the selection of alpha toxin mutation sites, changes of molecular structures before and after mutation, enzymatic properties and structural characteristics, and the relationship between the molecular structures and enzyme activities are not clear. The clostridium perfringens alpha toxin is a bacterial toxin without oxygen intolerance, and can hydrolyze lecithin to form phosphatidylcholine and insoluble diglyceride so as to cause hemolysis of red blood cells and decompose yolk phospholipid. Purified and concentrated clostridium perfringens alpha toxin can be prepared into aerosol as a biological weapon and can also be released in water and food, and the biological warfare agent can cause severe reactions of multiple systems and organs of human, such as anorexia, nausea and vomiting, stomach spasm and pain, purulent blood diarrhea; dyspnea, wheezing cough, oral cavity and throat pain, blood-stained sputum; burning, redness, swelling, itching, rash or blisters, and more seriously, death. Alpha toxin is the first bacterial protein found to have both enzymatic and toxic properties, and has a cold-heat hemolytic effect, i.e., when alpha toxin acts with erythrocytes at 37 ℃, erythrocytes are not lysed, and only when erythrocytes are cooled to 4 ℃, erythrocytes are lysed, which is found on mouse erythrocytes, rabbit erythrocytes, sheep erythrocytes, and horse erythrocytes. The alpha toxin is sensitive to pancreatin, and 2.5 percent of pancreatin and the alpha toxin can be completely inactivated after being acted for 1 hour at 37 ℃. In addition, alpha toxin can be inactivated by heating to 60-70 deg.C, and partial activity can be recovered by further heating to 100 deg.C. The size of the alpha toxin structural gene is 1194 bp, the coded product consists of 398 amino acids, of which 28 amino acids are signal peptides, and the rest 370 amino acids constitute mature protein, the relative molecular weight is 43000Da, and the isoelectric point pI is 5.1. The alpha toxin is divided into two structural domains, namely an N-terminal structural domain of an alpha helical structure and a C-terminal structural domain participating in membrane protein combination, and a large number of experiments prove that the N terminal and the C terminal of the alpha toxin have different activities, the N terminal has phospholipase C activity, the C terminal has sphingomyelinase activity, and only by the synergy of the two, the alpha toxin can have hemolytic activity and lethal activity. Alpha toxin is the most important pathogenic factor of clostridium perfringens, and no effective treatment method exists so far. The research on the humanized antibody against the alpha toxin is basically blank in China, and the research on the therapeutic antibody against the alpha toxin of the clostridium perfringens has wide application prospect.

A Single-chain antibody (Single-chain antibody), also called Single-chain antibody variable fragment (sc Fv), is formed by connecting an antibody heavy chain variable region (VH) and a light chain variable region (VL) via a flexible Linker. The molecular weight of the single-chain antibody is 27000-30000 Da, and the single-chain antibody is a functional structural unit which retains the whole antigen binding activity of the parent antibody to a small extent. The VH and VL of the antibody are folded to form the antibody with only one antigen binding site, and the antibody can only be specifically combined with one antigenic determinant. Currently, there are three main methods for preparing single-chain antibodies: (1) directly carrying out PCR amplification on a mixture of the heavy chain variable region, the light chain variable region and the connecting peptide to generate a single-chain antibody; (2) firstly, connecting VL and connecting peptide, and then connecting VH into sc Fv by PCR; (3) and respectively connecting the VH and the VL with a linker to form a VH-linker and a VL-linker, and carrying out PCR amplification on the VH upstream primer and the VL downstream primer which are provided with the enzyme cutting sites and are used for the rest amplification to form the scFv. It was found that single-chain antibodies formed by the differences in the direction and method of VH and VL linkage could be different, but the differences in the direction of VH and VL linkage did not significantly affect the specificity and affinity of single-chain antibodies, and that scFv with antigen binding capacity could be constructed, but the expression level of VL-VH in prokaryotic expression system was about 20 times higher than VH-VL.

At present, most of the preparation of humanized single-chain antibodies utilizes a phage display technology to construct a phage display library depending on a phage vector or a phagemid vector, a target antigen is specifically combined with the antibody, the required positive clone or target antibody is screened through several rounds of panning and enrichment processes, the qualitative and quantitative analysis is carried out on the positive clone or the target antibody by utilizing the technologies such as ELISA, PCR and the like, see Chinese patents ZL201410771046.3 and ZL201410771048.2 for details, the humanized single-chain antibody of the clostridium perfringens alpha toxin obtained by the patents is subjected to gene operation again, so that the humanized single-chain antibody is fused with a human IgG mFc fragment gene with a mutated hinge region, and the fully humanized single-chain antibody with good expression and preparation activity and stability is prepared.

Disclosure of Invention

The invention aims to overcome various side effects of a heterologous antibody and solve the problems of complicated steps and high cost of humanized transformation of the heterologous antibody, and provides a fully human ScFv-mFc monomolecular antibody of clostridium perfringens alpha toxin, which has small molecular weight and strong penetrating power in vivo, can quickly reach damaged tissues and cells to play an antitoxic role.

The base sequence of the fully human 7D-mFc monomolecular antibody for resisting clostridium perfringens alpha toxin is shown as SEQ ID NO. 1.

The amino acid sequence of the fully human 7D-mFc monomolecular antibody for resisting clostridium perfringens alpha toxin is shown as SEQ ID NO. 2.

Application of the fully human 7D-mFc monomolecular antibody for resisting clostridium perfringens alpha toxin in preparing a medicament for treating and preventing clostridium perfringens alpha toxin poisoning.

A detection kit for detecting clostridium perfringens alpha toxin comprises a clostridium perfringens alpha toxin fully human 7D-mFc monomolecular antibody with an amino acid sequence shown as SEQ ID NO. 2.

The invention provides a fully human 7D-mFc monomolecular antibody for resisting clostridium perfringens alpha toxin, and the base sequence of the antibody is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 2; the application of the fully human 7D-mFc monomolecular antibody for resisting the clostridium perfringens alpha toxin in preparing the medicine for treating and preventing the clostridium perfringens alpha toxin poisoning; a detection reagent for detecting clostridium perfringens alpha toxin comprises a clostridium perfringens alpha toxin fully human 7D-mFc monomolecular antibody with an amino acid sequence shown as SEQ ID NO. 2. The clostridium perfringens alpha toxin fully-humanized 7D-mFc single-molecule antibody is screened from a phage antibody library Source bioscience, is a fully-humanized anti-clostridium perfringens alpha toxin antibody, and is fused and expressed with a human IgG mFc fragment with a mutated hinge region through a genetic engineering technology, so that the treatment effect of neutralizing toxin can be realized, various side effects of a heterologous antibody are overcome, the complicated step and high cost of humanized modification of the heterologous antibody are avoided, the single-molecule antibody has small molecular weight and strong in vivo penetration capacity, quickly reaches damaged tissues and cells to play an antitoxin role, and after the human IgG Fc fragment is added, the in vivo stability is increased, the half-life is prolonged, and the in vivo clearance rate of the toxin can be improved, thereby achieving the dual purposes of economy and high efficiency. The alpha toxin has certain homology with other types of toxins of clostridium perfringens, the antibody drug has inhibition and treatment effects on other types of toxins, and can also be used as a detection reagent for detecting the clostridium perfringens alpha toxin. The hinge region mutated human IgG mFc fragment was mutated to cysteine in CPPCP and the hinge sequence was mutated to APPGP, intended to prevent intra-and intermolecular disulfide bond formation.

Drawings

FIG. 1 PCR and double restriction enzyme identification of recombinant expression plasmid P300-trxA-SUMO-7D-mFc; m: DL2000 DNAmarker; 1: recombining PCR amplification products of expression plasmid P300-trxA-SUMO-7D-mFc; 2: recombinant expression plasmids P300-trxA-SUMO-7D-mFc BamH I and EcoRI; 3: carrying out double-enzyme digestion electrophoresis on P300-trxA-SUMO-7D-mFc EcoR I and Xho I;

FIG. 2 shows the expression result of the engineering bacteria after induction; m: a protein Marker; 1: negative control; 2: inducing thalli;

FIG. 3 purified 7D-mFc protein; m is Marker; 1-7: 7D-mFc protein purification.

Detailed Description