阅读说明：本技术 抗H5N1病毒入胞抗体PTD-3F-mFc及其应用 (anti-H5N 1 virus entry antibody PTD-3F-mFc and application thereof ) 是由 张国利 高玉伟 刘楚含 田园 岳玉环 邓欣 吴广谋 李泽鸿 王铁成 刘雨玲 雍伟 于 2020-06-11 设计创作，主要内容包括：本发明公开了抗H5N1病毒入胞抗体PTD-3F-mFc,其碱基序列如序列表SEQID NO.7所示；制备方法为：按照人IgG的Fc基因序列设计突变铰链区半胱氨酸的CH2-CH3基因序列,并在各组分之间插入相应的内切酶酶切位点以方便后续基因操作,该设计基因序列进行人工合成,合成的基因连接入前期构建的PTS表达载体中,构建PTS-mFc表达载体；在此基础上再次克隆构建PTS-PTD-3F-mFc表达载体,将构建的表达载体转化入大肠杆菌中,诱导表达并纯化,比较其抗病毒活性,并优选单分子入胞抗体；以及本发明的单分子抗体在制备抗H5N1型人禽流感病毒药物的应用；结果表明PTD-3F-mFc抗H5N1病毒效价为600TCID50。(The invention discloses an anti-H5N 1 virus entry antibody PTD-3F-mFc, the base sequence of which is shown in a sequence table SEQID NO. 7; the preparation method comprises the following steps: designing a CH2-CH3 gene sequence of cysteine of a mutant hinge region according to an Fc gene sequence of human IgG, inserting corresponding endonuclease enzyme cutting sites among components to facilitate subsequent gene operation, artificially synthesizing the designed gene sequence, and continuously inserting the synthesized gene into a PTS expression vector constructed in the early stage to construct a PTS-mFc expression vector; on the basis, a PTS-PTD-3F-mFc expression vector is cloned and constructed again, the constructed expression vector is transformed into escherichia coli, induced expression and purification are carried out, the antiviral activity is compared, and a single molecule cell-entering antibody is optimized; the application of the monomolecular antibody in preparing the anti-H5N 1 type human avian influenza virus medicine is also disclosed; the results indicated that the PTD-3F-mFc anti-H5N 1 virus titer was 600TCID 50.)

1. The base sequence of the PTD-3F-mFc monomolecular antibody is shown as a sequence table SEQ ID NO. 7.

2. A method for preparing a PTD-3F-mFc monomolecular antibody, which comprises:

1) designing a CH2-CH3 gene sequence of cysteine of a mutant hinge region according to an Fc gene sequence of human IgG, inserting corresponding endonuclease enzyme cutting sites among components to facilitate subsequent gene operation, artificially synthesizing the designed gene sequence, and continuously inserting the synthesized gene into a PTS vector constructed in the early stage to construct a PTS-mFc expression vector; then, a PTD-3F fragment in the pET-28a-PTD-3F vector is amplified by utilizing PCR, PTD-3F is cloned into PTS-mFc, and a PTS-PTD-3F-mFc expression vector is constructed; the PTS carrier is a PET-TrxA-SUMO carrier which is constructed by self;

2) and transforming the constructed expression vector into escherichia coli, carrying out induced expression and purification, comparing the antiviral activity of the escherichia coli, and preferably selecting a single-molecule cell-entering antibody.

3. The PTD-3F-mFc monomolecular antibody is applied to the preparation of the anti-H5N 1 type human avian influenza virus medicine.

Technical Field

The invention belongs to the fields of bioengineering and prevention and treatment of major infectious diseases, and particularly relates to preparation of a fully human unimolecular cellular invasion antibody PTD-3F-mFc for resisting highly pathogenic avian influenza H5N1 virus, and application of a medicament for resisting H5N1 type human avian influenza virus.

Background

Human infection highly pathogenic avian influenza (abbreviated as human avian influenza) is a systemic or respiratory infectious disease caused by influenza A virus H5N1, and the fatality rate is more than 60%. Naturally, the host range of influenza virus infection has a certain specificity, and thus the virus can be divided into different groups, such as human influenza, avian influenza, swine influenza and the like, but the limit of the host range of influenza virus infection is not very strict, and the virus can be spread among different species. Since hong Kong reported human infection with H5N1 subtype avian influenza virus for the first time in 1997, highly pathogenic H5N1 avian influenza virus has continued to be epidemic in birds and human infection incidents have occurred, which has given brand-new public health significance to avian influenza, i.e. the great threat of highly pathogenic avian influenza virus to human health lies not only in causing current serious individual infection and even death, but also in the continuous variation of virus, possibly acquiring interpersonal transmission capability, resulting in a new round of pandemic of human influenza.

Under the condition that the virus generates drug resistance continuously, the novel antibody drug can become an effective means for coping with the potential influenza pandemic caused by the H5N1 virus. Currently, antiviral drugs are mainly used for treating human avian influenza, and currently approved antiviral drugs comprise two ion channel inhibitors and two neuraminidase inhibitors, but the influenza virus continuously generates drug resistance due to drug resistance-related site mutation and the like. If antiviral drugs are used for severe cases and critically ill patients for hyperactive symptoms for years and are commonly used as preventive drugs, the possibility of increasing drug resistance, high risk and serious public health safety consequences caused thereby cannot be excluded. In this case, it is necessary to prepare a novel anti-influenza virus drug. The antibody is very effective for treating severe influenza, but the heterologous antibody has strong immunogenicity, and is easy to cause severe allergic reaction in a human body in clinical application. With the development of genetic engineering technology, the development of genetic engineering antibodies is very rapid, wherein single-chain antibodies attract the attention of a plurality of researchers with the unique advantages of high specificity, small molecular weight, simple structure and low immunogenicity compared with parent antibodies, and can maximally reduce allergic reactions caused by foreign proteins in clinical application, the preparation technology thereof is mature, and particularly the phage display technology improves the screening efficiency of the antibodies and antibody genes. Therefore, single-chain antibodies will play an important role in the treatment of viral infectious diseases. However, the half-life of the single-chain antibody in vivo is short, generally about 25-30 minutes, which causes the virus neutralization effect in vivo to be incomplete, so the stability of the single-chain antibody in vivo must be increased, and the common method is to couple the single-chain antibody with other biological macromolecules or to fuse and express the single-chain antibody-biological macromolecules by genetic engineering.

The most important neutralizing antibody of the H5N1 virus is derived from the surface glycoprotein Hemagglutinin (HA), so HA HAs been the main target of previous research. However, the H5N1 virus HAs high variability, and the prediction result according to molecular evolution tree analysis shows that the HA gene of the H5N1 virus HAs evolved into at least 10 variant branches with different antigenic characteristics, and the immunological cross reaction between the different branches is weaker. Due to the influence of virus variation, the antibody based on the conserved antigen component can generate antiviral effect on different subtype avian influenza viruses. The influenza virus matrix protein M1 is the main structural protein of avian influenza virus, is located inside the virus envelope, and participates in and regulates the processes of virus replication, transcription, release and the like by combining with host cell target protein. The M1 protein sequence is conserved, so that the antibody aiming at M1 can inhibit the activity thereof by combining with M1 protein, interfere the replication, transcription and release of each subtype avian influenza virus, and play a role in broad-spectrum virus resistance.

The cell-entering antibody based on the protein transduction domain can enter cells, and is combined with an intracellular target antigen to exert biological activity. The M1 protein antibody can generate antiviral activity to various subtype avian influenza viruses, but H5N1 virus is replicated and packaged in cells, and M1 protein is located on the inner side of a virus envelope, so that the antibody can play an antiviral role only after entering infected cells. Protein Transduction Domains (PTDs) are small peptide fragments that mediate proteins across cell membranes, carrying macromolecules efficiently across the biological membrane into the cell. PTD-mediated protein transport does not depend on receptor, channel, energy and endocytosis, can directly act on lipid bilayers of all types of cells to complete transmembrane movement, and has no species specificity in transmembrane function. Since the identification and characterization of PTDs, hundreds of compounds and proteins have been successfully used for transduction, carrying biological macromolecules into diverse cells and exhibiting corresponding biological activities. Among the PTDs discovered, the PTD of the TAT protein of human immunodeficiency virus-1 (HIV-1) is the most studied and well-defined function, and the PTD of the TAT protein can transfer the connected polypeptide, protein and DNA into cells efficiently and quickly in a concentration-dependent manner, while the normal structure and function of the cells are not influenced. Although the mechanism of protein transduction is currently under study, the property of directly delivering therapeutic biomacromolecules into cells to exert biological effect provides a new idea for biological treatment of diseases, and thus the protein transduction mechanism is widely concerned in the field of medical research. In 1997, Vives et al found that the PTD of TAT is 11 amino acids located at positions 47-57 (YGRKKRRQRRR), which is a polypeptide fragment rich in basic amino acids. To increase the transduction efficiency of this PTD, its second Gly was mutated to a hydrophobic His. In view of the biological transduction characteristic of TAT PTD, after the fragment is fused and expressed with ScFv or ScFv-mFc of anti-M1 protein, the fragment can bring anti-M1 antibody components into virus infected cells, target M1 protein in cells, prevent the M1 antibody components from playing biological functions, inhibit the assembly and release of influenza virus, and play a role in resisting virus.

Disclosure of Invention

The invention aims to provide a preparation method and application of a fully human single-molecule cellular-in antibody PTD-3F-Fc for resisting highly pathogenic avian influenza H5N1 virus.

The base sequence of the PTD-3F-mFc monomolecular antibody is shown as a sequence table SEQ ID NO. 7.

A method for preparing a PTD-3F-mFc monomolecular antibody, which comprises:

1) designing a CH2-CH3 gene sequence of cysteine of a mutant hinge region according to an Fc gene sequence of human IgG, inserting corresponding endonuclease enzyme cutting sites among components to facilitate subsequent gene operation, artificially synthesizing the designed gene sequence, connecting the synthesized gene into a PTS vector constructed in the early stage to construct a PTS-mFc expression vector, cloning a PTD-3F gene fragment into the PTS-mFc to construct the PTS-PTD-3F-mFc expression vector; the PTS carrier is a PET-TrxA-SUMO carrier which is constructed by self;

2) and transforming the constructed expression vector into escherichia coli, carrying out induced expression and purification, comparing the antiviral activity of the escherichia coli, and preferably selecting a single-molecule cell-entering antibody.

The PTD-3F-mFc monomolecular antibody is applied to the preparation of the anti-H5N 1 type human avian influenza virus medicine.

The invention provides a PTD-3F-mFc monomolecular antibody, the base sequence of which is shown as a sequence table SEQ ID NO. 7; the preparation method comprises the following steps: designing a CH2-CH3 gene sequence of cysteine of a mutant hinge region according to an Fc gene sequence of human IgG, inserting corresponding endonuclease enzyme cutting sites among components to facilitate subsequent gene operation, artificially synthesizing the designed gene sequence, connecting the synthesized gene into a PTS vector constructed in the early stage to construct a PTS-mFc expression vector, cloning a PTD-3F gene fragment into the PTS-mFc to construct the PTS-PTD-3F-mFc expression vector; the PTS carrier is a PET-TrxA-SUMO carrier which is constructed by self; transforming the constructed expression vector into escherichia coli, performing induced expression and purification, comparing antiviral activity of the escherichia coli, and preferably selecting a single-molecule cell-entering antibody; the PTD-3F-mFc monomolecular antibody is applied to the preparation of the anti-H5N 1 type human avian influenza virus medicine. The invention selects human highly pathogenic avian influenza virus H5N1 conserved sequence M1 protein as target antigen, and utilizes phage antibody library to screen out human high affinity single-chain antibody of M1 protein; in order to increase the transduction efficiency of YGRKKRRQRRR, the Gly of the second cis position is mutated into His to increase the hydrophobicity and the transport rate, and the mutated TAT protein PTD is connected with the anti-M1 protein single-chain antibody gene to construct and express the PTD-ScFv. To increase its stability in vivo, PTD-ScFv was expressed in fusion with an Fc fragment of human IgG with a mutated hinge region: the fusion protein is PTD-ScFv-CH2-CH3, wherein the hinge region mutation Fc is that cysteine in CPPCP is mutated into APPGP, which is intended to prevent the formation of disulfide bond in and among molecules, thereby preparing the fully human single molecule cell-entering antibody of anti-human avian influenza virus; the results indicated that the PTD-3F-mFc titer was 600TCID 50.

Drawings

FIG. 1 shows the expression result of the engineering bacteria for expressing the PET-SUMO-M1 protein after induction; m: a protein Marker;1: negative control; 2: inducing the ultrasonic precipitation of thalli; 3: inducing the thallus to carry out ultrasonic supernatant; 4: inducing the whole bacteria;

FIG. 2 purified M1 protein; m is Marker, 1 is M1 sample after SUMO enzyme digestion and purification;

FIG. 3 amplification of the PTD-ScFv gene; m is Marker;1: PTD-7B PCR product, 2: PTD-3F

FIG. 4 purified PTD-3F; m is Marker, 1 is PTD-3F;

FIG. 5 shows the induction expression result of PTS-PTD-3F-mFc/BL 21(DE3) in recombinant expression strain; m: protein molecular weight Marker; 1. 3, negative control; 2, inducing the recombinant expression vector PTS-PTD-7B-mFc; 4: inducing a recombinant expression vector PTS-PTD-3F-mFc;

FIG. 6 electrophoretogram of purified PTD-3F-mFc protein; m: protein molecular weight Marker;1: pre-molecular sieve PTD-3F-mFc protein as such, 2-5: for isolated hetero-protein 6: PTD-3F-mFc protein purified samples.

Detailed Description