Single chain antibody for resisting SARS-COV-2 virus S protein and its use
阅读说明：本技术 抗sars-cov-2病毒s蛋白的单链抗体及其用途 (Single chain antibody for resisting SARS-COV-2 virus S protein and its use ) 是由 罗绍祥 易汪雪 张芳 王静 于 2020-07-02 设计创作，主要内容包括：本发明提供了一种抗SARS-COV-2病毒S蛋白的单链抗体,所述单链抗体包括单链抗体1和单链抗体2中的至少一种：单链抗体1：具有如SEQ ID NO：1-SEQ ID NO：3所示的氨基酸序列的三个互补决定区的重链和具有如SEQ ID NO：4-SEQ ID NO：6所示的氨基酸序列的三个互补决定区的轻链；单链抗体2：具有如SEQ ID NO：7-SEQ ID NO：9所示的氨基酸序列的三个互补决定区的重链和具有如SEQ ID NO：10-SEQ ID NO：12所示的氨基酸序列的三个互补决定区的轻链。本发明还提供所述单链抗体在制备SARS-COV-2病毒检测试试剂盒中的用途。(The invention provides a single-chain antibody for resisting SARS-COV-2 virus S protein, the single-chain antibody comprises at least one of single-chain antibody 1 and single-chain antibody 2: single-chain antibody 1: has the sequence shown in SEQ ID NO: 1-SEQ ID NO: 3 and a light chain having three complementarity determining regions of the amino acid sequence set forth in SEQ ID NO: 4-SEQ ID NO: 6, and a light chain of three complementarity determining regions of the amino acid sequence set forth in seq id no; single-chain antibody 2: has the sequence shown in SEQ ID NO: 7-SEQ ID NO: 9 and a light chain having three complementarity determining regions of the amino acid sequence set forth in SEQ ID NO: 10-SEQ ID NO: 12, or a light chain of three complementarity determining regions of the amino acid sequence set forth in seq id no. The invention also provides the use of the single-chain antibody in the preparation of a SARS-COV-2 virus detection reagent kit.)
1. A single chain antibody against the S protein of SARS-COV-2 virus, said single chain antibody recognizing the RBD domain of the S protein of SARS-COV-2 virus, said single chain antibody comprising at least one of single chain antibody 1 and single chain antibody 2:
Single-chain antibody 1: comprises a polypeptide with the sequence shown in SEQ ID NO: 1-SEQ ID NO: 3 and a light chain having three complementarity determining regions of the amino acid sequence set forth in SEQ ID NO: 4-SEQ ID NO: 6, and a light chain of three complementarity determining regions of the amino acid sequence set forth in seq id no;
single-chain antibody 2: comprises a polypeptide with the sequence shown in SEQ ID NO: 7-SEQ ID NO: 9 and a light chain having three complementarity determining regions of the amino acid sequence set forth in SEQ ID NO: 10-SEQ ID NO: 12, or a light chain of three complementarity determining regions of the amino acid sequence set forth in seq id no.
2. The single chain antibody against SARS-COV-2 virus S protein of claim 1, wherein the single chain antibody comprises one or more of the following single chain antibodies:
h6 single chain antibody: comprises a polypeptide with the sequence shown in SEQ ID NO: 13 and a light chain having the amino acid sequence set forth in SEQ ID NO: 14, a light chain of an amino acid sequence set forth in seq id no;
d2 single chain antibody: comprises a polypeptide with the sequence shown in SEQ ID NO: 15 and a light chain having the amino acid sequence set forth in SEQ ID NO: 16, or a light chain of the amino acid sequence set forth in seq id no.
3. The single-chain antibody against SARS-COV-2 virus S protein according to claim 2, wherein the single-chain antibody against SARS-COV-2 virus S protein further comprises: the H6 single-chain antibody and the D2 single-chain antibody have the same functions obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequences.
4. The single-chain antibody against SARS-COV-2 virus S protein according to claim 2 or 3, wherein the single-chain antibody against SARS-COV-2 virus S protein further comprises: the H6 single-chain antibody and the D2 single-chain antibody are obtained by connecting labels at the N end and/or the C end.
5. A nucleic acid molecule encoding the single chain antibody of any one of claims 1 to 4.
6. The nucleic acid molecule according to claim 5, wherein said nucleic acid molecule comprises a nucleotide molecule encoding said single-chain antibody 1 and/or a nucleotide molecule encoding said single-chain antibody 2.
7. A biological material comprising a nucleic acid molecule according to any one of claims 5 to 6, wherein the biological material comprises recombinant DNA, a plasmid vector, a phage vector, a viral vector, an engineered bacterium or a transgenic cell line.
8. A recombinant antibody, which is composed of the single-chain antibody against SARS-COV-2 virus S protein according to any one of claims 1 to 4 and a human Fc fragment, wherein the amino acid sequence of the human Fc fragment is as shown in SEQ ID NO: shown at 21.
9. The use of the single-chain antibody of the S protein RBD domain of SARS-COV-2 virus as claimed in any of claims 1-4 and the recombinant antibody as claimed in claim 8 in the preparation of medicaments for inhibiting SARS-COV-2 virus infection, and in the preparation of SARS-COV-2 virus detection test reagents or kits.
10. A reagent or a kit for detecting SARS-COV-2 virus, comprising a single-chain antibody of the RBD domain of the S protein of SARS-COV-2 virus according to any one of claims 1 to 4 or a recombinant antibody according to claim 8.
Technical Field
The present invention belongs to the field of biotechnology, and relates to a single chain antibody of SARS-COV-2 virus S protein and its application.
Background
Coronavirus is a single-stranded RNA virus that is hosted by mammals and birds. Coronaviruses that can infect humans include 229E, NL63, OC43, HKU1, SARS-CoV and MERS-CoV. These viruses can cause a range of mild seasonal illnesses, or cause severe disease outbreaks, most typically Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) caused by SARS-CoV and MERS-CoV. SARS-CoV-2 is a seventh coronavirus capable of infecting humans, which is more contagious than SARS-CoV and MERS-CoV, resulting in a broad outbreak of COVID-19.
Coronaviruses consist of a bilayer lipid envelope containing spike, envelope, membrane and nucleocapsid proteins. Among them, the membrane protein mediates the interaction of the virus with the host ACE2 protein, and SARS-CoV-2 and SARS-CoV use the same invasion receptor. The sequence alignment shows that the amino acid sequence homology of SARS-CoV-2 and SARS-CoV is 80%. The S protein is divided into two subunits of S1 and S2, wherein the function of S1 is to bind to host receptors, and the main function of S2 is to promote fusion of virus and cell membrane. The high affinity of the RBD domain of S1 for binding to ACE2 protein results in rapid spread of the virus in the human population. The S protein is exposed on the surface of the virus, contains a large number of antigenic determinants and can generate protective antibodies against the virus. Therefore, the S protein is the most important target for disease diagnosis and treatment.
The single chain antibody (scFv) is formed by connecting an antibody heavy chain variable region and an antibody light chain variable region through a short peptide (linker) with 15-20 amino acids. Advantages of scFv Single chain antibodies: competitive surface protein of non-specific reaction can be removed, and the background of tumor visualization is clearer; the immunity is small, and the rejection reaction of human anti-mouse can be eliminated; the half-life period of the circulation in the body is short, the clearing is easy, and the detoxification and the discharge are facilitated; is easy to connect with toxin or enzyme gene, and is convenient for directly obtaining immunotoxin or enzyme-labeled antibody, etc.
Therefore, there is a strong need to develop new single chain antibodies for SARS-COV-2.
Disclosure of Invention
In order to solve the technical problem, the invention provides a single-chain antibody of SARS-COV-2 virus S protein and the application thereof, which can specifically identify RBD protein, and the EC50 of the single-chain antibody combined with the RBD is 0.4212 ng/ml-325.24 ng/ml.
In a first aspect, the present invention provides a single chain antibody against SARS-COV-2 virus S protein, which single chain antibody recognizes the RBD domain of SARS-COV-2 virus S protein, the single chain antibody comprising at least one of
single-chain antibody 1: comprises a polypeptide with the sequence shown in SEQ ID NO: 1-SEQ ID NO: 3 and a light chain having three complementarity determining regions of the amino acid sequence set forth in SEQ ID NO: 4-SEQ ID NO: 6, and a light chain of three complementarity determining regions of the amino acid sequence set forth in seq id no;
single-chain antibody 2: comprises a polypeptide with the sequence shown in SEQ ID NO: 7-SEQ ID NO: 9 and a light chain having three complementarity determining regions of the amino acid sequence set forth in SEQ ID NO: 10-SEQ ID NO: 12, or a light chain of three complementarity determining regions of the amino acid sequence set forth in seq id no.
Further, the single chain antibody includes one or more of the following single chain antibodies:
H6 single chain antibody: comprises a polypeptide with the sequence shown in SEQ ID NO: 13 and a light chain having the amino acid sequence shown as SEQ id no: 14, a light chain of an amino acid sequence set forth in seq id no;
d2 single chain antibody: comprises a polypeptide with the sequence shown in SEQ ID NO: 15 and a light chain having the amino acid sequence shown in SEQ id no: 16, or a light chain of the amino acid sequence set forth in seq id no.
Further, the single chain antibody against SARS-COV-2 virus S protein further comprises: the H6 single-chain antibody and the D2 single-chain antibody have the same functions obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequences.
Further, the single chain antibody against SARS-COV-2 virus S protein further comprises: the H6 single-chain antibody and the D2 single-chain antibody are obtained by connecting labels at the N end and/or the C end.
In a second aspect, the invention provides a nucleic acid molecule encoding said single chain antibody.
Further, the nucleic acid molecule includes a nucleotide molecule encoding the single-
Further, the nucleotide sequence of the nucleotide molecule encoding the heavy chain of the single-
In a third aspect, the invention provides a biological material containing the nucleic acid molecule, wherein the biological material comprises recombinant DNA, a plasmid vector, a phage vector, a viral vector, an engineered bacterium or a transgenic cell line.
In a fourth aspect, the present invention provides a recombinant antibody, wherein the recombinant antibody comprises a single chain antibody against SARS-COV-2 virus S protein and a human Fc fragment, and the amino acid sequence of the human Fc fragment is as shown in SEQ ID NO: shown at 21.
In the fifth aspect, the invention provides the single chain antibody of the S protein RBD structural domain of the SARS-COV-2 virus and the application of the recombinant antibody in the preparation of drugs for inhibiting SARS-COV-2 virus infection, and the preparation of SARS-COV-2 virus reagents or kits.
In the sixth aspect, the invention provides a SARS-COV-2 virus detection reagent or kit, comprising the single-chain antibody of the S protein RBD structural domain of the SARS-COV-2 virus or the recombinant antibody.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the single-chain antibody of the S protein of the SARS-COV-2 virus provided by the invention has the EC50 combined with the S1 protein of 42.83ng/ml and 245.13ng/ml respectively; the EC50 of the single-chain antibody combined with the RBD protein is 29.51ng/ml and 152.45ng/ml respectively; the single-chain antibody competes with a virus receptor ACE2 protein, IC50 is 23.32ng/ml and 125.45ng/ml respectively, and the single-chain antibody has the capacity of inhibiting virus infection. The SARS-COV-2 virus detection kit provided by the invention comprises the single-chain antibody or the recombinant antibody composed of the single-chain antibody and the humanized Fc fragment, and has strong specificity and high sensitivity.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a graph of the binding of the recombinant H6 antibody to the S1 protein in example 3;
FIG. 2 is a graph showing the binding of H6 recombinant antibody to RBD protein in example 4;
FIG. 3 is the competition of the H6 recombinant antibody with ACE2 protein in example 5;
FIG. 4 shows the sensitivity of the single-chain antibody when applied to a colloidal gold assay kit.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
the S protein is exposed on the surface of the virus, contains a large number of antigenic determinants, can generate protective antibodies against the virus, and is a main antigen for vaccine development and neutralizing antibody development. Meanwhile, due to the strong immunogenicity, a large amount of antibodies can be generated in the virus body, and the antibody is also an important raw material for developing a diagnostic kit.
The natural antibody library is suitable for screening various antigens, a mouse is not required to be singly immunized, and the antibody for identifying the target protein can be screened within one week by combining a high-throughput screening scheme of a phage display technology. This study used recombinant expressed S protein to screen the natural antibody library of mice, and obtained 2 strains of single chain antibodies that can specifically recognize S protein:
h6 single chain antibody: comprises a polypeptide with the sequence shown in SEQ ID NO: 13 and a light chain having the amino acid sequence shown as SEQ id no: 14, a light chain of an amino acid sequence set forth in seq id no; the heavy chain has the amino acid sequence of SEQ ID NO: 1-SEQ ID NO: 3, and the light chain has the amino acid sequence set forth in SEQ ID NO: 4-SEQ ID NO: 6, and (b) three complementarity determining regions of the amino acid sequence set forth in figure 6.
D2 single chain antibody: comprises a polypeptide with the sequence shown in SEQ ID NO: 15 and a light chain having the amino acid sequence shown in SEQ id no: 16, or a light chain of the amino acid sequence set forth in seq id no. The heavy chain has the amino acid sequence of SEQ ID NO: 7-SEQ ID NO: 9, and the light chain has the amino acid sequence shown in SEQ ID NO: 10-SEQ ID NO: 12, or a sequence of three complementarity determining regions of the amino acid sequence set forth in seq id no.
The amino acid sequences of the CDR regions of the single chain antibody are shown in table 1 below:
TABLE 1
Therefore, single-chain antibodies having the heavy chain complementarity determining regions CDR1-CDR3 and light chain complementarity determining regions CDR1-CDR3 of the H6 single-chain antibody, and single-chain antibodies having the heavy chain complementarity determining regions CDR1-CDR3 and light chain complementarity determining regions CDR1-CDR3 of the D2 single-chain antibody are also within the scope of the present invention, i.e., the single-chain antibody 1-single-
The single-chain antibodies can be specifically combined with an RBD region of an S protein of a novel coronavirus SARS-COV-2 through experimental verification, and EC50 of the combination of an H6 recombinant antibody and a D2 recombinant antibody with an S1 protein is 42.83ng/ml and 245.13ng/ml respectively; the EC50 of the H6 recombinant antibody and the D2 recombinant antibody combined with the RBD protein is 29.51ng/ml and 152.45ng/ml respectively. The H6 recombinant antibody and the D2 recombinant antibody compete with the virus receptor ACE2 protein, IC50 is 23.32ng/ml and 125.45ng/ml respectively, and the antibodies have the capacity of inhibiting virus infection.
In addition, the invention also relates to a recombinant antibody obtained after recombining the single-chain antibody;
the single-chain antibody or the recombinant antibody is used for a colloidal gold detection kit of SARS-COV-2 virus, and has strong specificity, high accuracy and high sensitivity.
The effects of the present application will be described in detail below with reference to examples and experimental data.