Hybridoma cell strain secreting tomato ringspot virus monoclonal antibody, antibody thereof and antibody preparation method
阅读说明：本技术 分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株及其抗体和抗体制备方法 (Hybridoma cell strain secreting tomato ringspot virus monoclonal antibody, antibody thereof and antibody preparation method ) 是由 张蓬军 俞晓平 孙凯 杨倩倩 刘光富 许益鹏 王正亮 于 2019-11-20 设计创作，主要内容包括：本发明提供一种分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株及其抗体和抗体制备方法,其特征在于：以抗原肽CAFGKGVEEIEQTST免疫BALB/c小鼠,获得杂交瘤细胞株,并从中筛选出能够产生特异性识别该抗原肽单克隆抗体的细胞株,并通过腹水制备法获得特异性识别番茄环斑病毒外壳蛋白AFGKGVEEIEQTST肽段的单克隆抗体,该单克隆抗体重链可变区和轻链可变区长度分别为185个和103个氨基酸；单克隆抗体的产量为500-5000mg/L腹水,纯度为85％-99.5％。本发明单克隆抗体具有特异性识别番茄环斑病毒外壳蛋白的特性,在番茄环斑病毒进出口检验检疫方面具有广阔应用前景。(The invention provides a hybridoma cell strain secreting monoclonal antibody of tomato ringspot virus, an antibody thereof and a preparation method of the antibody, which is characterized in that: immunizing a BALB/c mouse by using an antigenic peptide CAFGKGVEEIEQTST to obtain a hybridoma cell strain, screening a cell strain capable of generating a monoclonal antibody capable of specifically recognizing the antigenic peptide from the hybridoma cell strain, and obtaining a monoclonal antibody capable of specifically recognizing a tomato ringspot virus coat protein AFGKGVEEIEQTST peptide segment by an ascites preparation method, wherein the lengths of a heavy chain variable region and a light chain variable region of the monoclonal antibody are 185 amino acids and 103 amino acids respectively; the yield of the monoclonal antibody is 500-5000mg/L ascites, and the purity is 85-99.5%. The monoclonal antibody has the characteristic of specifically recognizing the coat protein of the tomato ringspot virus, and has wide application prospect in the aspects of import and export inspection and quarantine of the tomato ringspot virus.)
1. A tomato ringspot virus monoclonal antibody is characterized in that: the variable region of the heavy chain of the antibody has a sequence shown in SEQ ID NO: 1, the light chain variable region sequence is SEQ ID NO: 2, respectively.
2. The monoclonal antibody of claim 1, wherein the monoclonal antibody heavy chain variable region and light chain variable region are 185 and 103 amino acids in length, respectively.
3. A gene encoding the monoclonal antibody of claim 1, comprising a nucleotide sequence encoding a light chain variable region and a nucleotide sequence encoding a heavy chain variable region, the nucleotide sequence encoding the heavy chain variable region being as set forth in SEQ id no: 3, and the nucleotide sequence for coding the light chain variable region is shown as SEQ ID NO: 4, respectively.
4. The hybridoma cell strain capable of secreting the tomato ringspot virus monoclonal antibody is characterized by being preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation numbers are as follows: CGMCC No. 18320.
The 185 amino acid residue sequence of the heavy chain variable region was analyzed by BLASTP and the first matched sequence similarity was 89.77%; the 103 amino acid residue sequence of the light chain was analyzed by BLASTP for first matched sequence similarity of 56.14%.
5. A method for producing the monoclonal antibody of claim 1, characterized by comprising the steps of:
(1) injecting 0.1-0.5mL of sterilized paraffin into the abdominal cavity of a BALB/c mouse for 8-15 weeks;
(2) intraperitoneal inoculation of 5-10 days later, hybridoma cells of claim 4 suspended in PBS, 5 x 105-5×106A/only;
(3) collecting ascites 2-4 times after 5-10 days;
(4) centrifuging 1000 Xg ascites for 10min, sucking upmost adipose tissue, removing cell components and precipitate, and collecting supernatant;
(5) 3mL of ascites supernatant is taken, added with 3mL of 0.06M sodium acetate-acetic acid buffer solution with the pH value of 4.0 to 4.6; adding 99uL n-octanoic acid dropwise, stirring at 0-10 deg.C for 10-60min, and clarifying for 10-60 min; centrifuging at 14000 Xg for 20min at 4 deg.C, removing precipitate, adding 1/10 volume of 10 XPBS into supernatant, and adjusting pH to 7.2 with 1M NaOH; dropwise adding equivalent saturated ammonium sulfate solution into the supernatant, continuously stirring for 30min, and standing for 30 min; centrifuging at 14000 Xg for 30min at 4 deg.C, discarding the supernatant, dissolving the precipitate in 1.2mL PBS, dialyzing with 0.01M, pH7.4 PBS at 0-10 deg.C overnight, and concentrating with 5% PEG20000 to 5 mL;
(6) and (3) passing the concentrated solution through protein G purification resin, washing 10-15 column volumes by using pre-elution solution, eluting the monoclonal antibody by using 5-10 column volumes of elution buffer solution, and concentrating to obtain the monoclonal antibody.
6. The method according to claim 5, wherein the pre-elution solution in the step (6) comprises 0.1 to 0.15M NaCl and 10 to 40mM Na2HPO4pH 7.0; the elution buffer solution is 0.1-0.3M glycine, and the pH value is 3.0.
7. The use of the tomato ringspot virus monoclonal antibody of claim 1 in the preparation of a tomato ringspot virus detection reagent or kit.
Technical Field
The invention relates to a monoclonal antibody, in particular to a hybridoma cell strain secreting a tomato ringspot virus monoclonal antibody, an antibody thereof and an antibody preparation method.
Background
Tomato ringspot virus (ToRSV) is a member of the genus Nepovirus (Comoviridae) of the Comoviridae family, and the virus particles are isodiametric icosahedron with a diameter of about 30 nm. The genome contains two positive-sense single-stranded RNAs which respectively code for replicase, Motor Protein (MP) and Coat Protein (CP). ToRSV is widely distributed in Europe, America and the state of the America, can infect more than 150 crops such as soybean, grape, peach, plum, apple, cherry, tomato, tobacco and the like, causes various diseases and leads to outcropping in serious cases. There are several modes of ToRSV transmission, and the vectors of the nematode (Xiphinema) and graft transmission in the field are important modes of ToRSV transmission and prevalence, while long-distance transmission is mainly based on seed (seedling) regulation. The seed-borne poison plants are many, and some have high seed-borne rate, such as 76% of soybean, 76% of globe amaranth, 11% of elderberry, 24% of dandelion, 3-7% of red clover and 3% of tomato. The virus is a high-risk virus and is a quarantine pest prohibited from entry in China. Since ToRSV is a high-risk quarantine harmful organism which can be remotely transmitted along with the transportation of seed seedlings, establishing an effective rapid detection method is very important for preventing the virus from entering China. At present, the means used for inspection and quarantine against ToRSV at home and abroad include electron microscope observation, differential host reaction (biological method), serological test and molecular biological means. Since ToRSV is a spherical virus with the diameter of 28nm, the virus content in a sample is low, and the virus is difficult to be observed and identified under an electron microscope; the inoculation of the differential hosts requires a special isolation greenhouse, takes a long time and influences the identification result due to the occult phenomenon of viruses. The serology method is a necessary means for rapidly detecting the plant virus, the virus detection of the port depends on antiserum purchased abroad at present, the price is high, and the ToRSV serum detection kit for the imported plant seedlings is not reported at home.
Aiming at the current situation that a large number of seedlings imported in China possibly carry tomato ringspot virus, the invention screens and obtains a monoclonal antibody cell strain by taking tomato ringspot virus coat protein as a detection target through the selection and synthesis of a tomato ringspot virus specific universal antigen peptide segment, cross-linking immune mice, cell fusion, antibody activity detection and other series of steps. And obtaining antigen recognition region sequences in the antibody gene, namely heavy chain variable region sequences and light chain variable region sequences by an RT-PCR method. Finally obtaining the monoclonal antibody with known sequence for specifically recognizing the target antigen, the cell strain for producing the antibody and the corresponding antibody production method. Compared with the method for obtaining the monoclonal antibody by immunizing a mouse with the purified virus particles, the monoclonal antibody obtained by the invention has definite recognition epitope, and the monoclonal antibody obtained by immunizing the virus particles has indefinite recognition epitope. In addition, the invention firstly separates out a conserved region from the tomato ringspot virus coat protein sequence, then selects and designs a candidate antigen peptide segment from the conserved region, and then adopts a chemical synthesis method to prepare the antigen peptide segment, so that the acquisition of the antigen is not limited by a source of a virus-carrying sample, and the operation permission range of an imported inspection and quarantine substance is not violated. The specific recognition epitope also enables the monoclonal antibody of the invention to have specific and easily recognized characteristics with the existing antibodies on the market and the related research report antibodies.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a tomato ringspot virus monoclonal antibody which has the reaction specificity of identifying and combining the specific peptide segment of the tomato ringspot virus coat protein and lacks the combination specificity to other viruses and plant tissues, thereby laying a foundation for further developing and developing a tomato ringspot virus serum detection kit.
The invention firstly discloses a tomato ringspot virus monoclonal antibody, wherein the sequence of a heavy chain variable region of the antibody is SEQ ID NO: 1, the light chain variable region sequence is SEQ ID NO: 2, respectively.
The length of the heavy chain variable region and the length of the light chain variable region of the monoclonal antibody are 185 and 103 amino acids respectively.
As a preferred embodiment of the present invention, the nucleotide sequences encoding the heavy chain and light chain variable regions are SEQ id nos: 3 and SEQ ID NO: 4, respectively. The 185 amino acid residue sequence of the heavy chain variable region was analyzed by BLASTP and the first matched sequence similarity was 89.77%; the 103 amino acid residue sequence of the light chain was analyzed by BLASTP for first matched sequence similarity of 56.14%.
The monoclonal antibody is prepared by an ascites induction method, the yield is 500-5000mg/L, and the purity is 85-99.5%.
The invention also discloses a hybridoma cell strain (mouse hybridoma cell 5H9H10) for secreting the tomato ringspot virus monoclonal antibody, which is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation numbers are as follows: CGMCC No. 18320. The preservation date is 8 months and 15 days in 2019, and the address of a preservation unit is No. 3 Xilu No.1 on Beijing, Chaoyang district.
The invention also discloses application of the tomato ringspot virus monoclonal antibody in preparation of a tomato ringspot virus detection reagent or kit.
The tomato ringspot virus monoclonal antibody is prepared by the following steps:
(1) searching and obtaining a plurality of coat protein sequences (including protein sequences translated by gene sequences) of tomato ringspot viruses from Genbank, and selecting conserved segments from the coat protein sequences by sequence comparison;
(2) selecting a proper antigen sequence aiming at the conserved segment to obtain an optimized antigen peptide segment CAFGKGVEEIEQTST which is shown as SEQ ID NO. 5;
(3) synthesizing the antigen peptide segment CAFGKGVEEIEQTST with 1 cysteine residue added at the N terminal by a chemical synthesis method;
(4) crosslinking the antigen peptide segment with KLH;
(5) immunizing a mouse with the cross-linked antigenic peptide fragment;
(6) preparing hybridoma cells and screening cell strains which produce antibodies which specifically recognize the peptide fragments of SEQ ID NO. 5 from the hybridoma cells;
(7) adopting an RT-PCR method to clone and measure the variable region sequences of the heavy chain and the light chain of the monoclonal antibody in the monoclonal antibody cell strain;
(8) preparing a monoclonal antibody which can identify the peptide segment of SEQ ID NO. 5 and make positive reaction on a sample carrying the tomato ringspot virus by adopting an ascites induction method;
(9) the monoclonal antibody of the invention is purified by ammonium sulfate precipitation and protein G affinity layer washing.
The preparation process of the ascites induction method of the monoclonal antibody specifically comprises the following steps:
(1) injecting 0.1-0.5mL of sterilized paraffin into the abdominal cavity of a BALB/c mouse for 8-15 weeks;
(2)5-10 days later, the hybridoma cells were inoculated intraperitoneally with 5X 10 of PBS suspension5-5×106A/only;
(3) collecting ascites 2-4 times after 5-10 days;
(4) centrifuging 1000 Xg ascites for 10min, sucking upmost adipose tissue, removing cell components and precipitate, and collecting supernatant;
(5) 3mL of ascites supernatant is taken, added with 3mL of 0.06M sodium acetate-acetic acid buffer solution with the pH value of 4.0 to 4.6; adding 99uL n-octanoic acid dropwise, stirring at 0-10 deg.C for 10-60min, and clarifying for 10-60 min; centrifuging at 14000 Xg for 20min at 4 deg.C, removing precipitate, adding 1/10 volume of 10 XPBS into supernatant, and adjusting pH to 7.2 with 1M NaOH; dropwise adding equivalent saturated ammonium sulfate solution into the supernatant, continuously stirring for 30min, and standing for 30 min; centrifuging at 14000 Xg for 30min at 4 deg.C, discarding the supernatant, dissolving the precipitate in 1.2mL PBS, dialyzing with 0.01M, pH7.4 PBS at 0-10 deg.C overnight, and concentrating with 5% PEG20000 to 5 mL;
(6) and (3) passing the concentrated solution through protein G purification resin, washing 10-15 column volumes by using pre-elution solution, eluting the monoclonal antibody by using 5-10 column volumes of elution buffer solution, and concentrating to obtain the monoclonal antibody.
Preferably, the pre-elution solution in step (6) comprises 0.1-0.15M NaCl and 10-40mM Na2HPO4pH 7.0; the elution buffer solution is 0.1-0.3M glycine, and the pH value is 3.0.
The term "monoclonal antibody (mab)" as used herein refers to an antibody obtained from a substantially homogeneous population of cells, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. The monoclonal antibody specifically recognizes a peptide segment shown in SEQ ID NO. 5 derived from tomato ringspot virus coat protein, has a unique and definite recognition sequence, is different from a conventional monoclonal antibody with an unknown recognition sequence obtained by immunizing a mouse with tomato ringspot virus, and is also definitely different from a tomato ringspot virus polyclonal antibody.
The invention has the following beneficial effects: the results of potency determination and enzyme-linked immunoassay of the tomato ringspot virus monoclonal antibody show that the monoclonal antibody for identifying the peptide segment shown in SEQ ID NO. 5 has the specificity of reaction with tomato ringspot virus, can be used as an antibody for detecting the tomato ringspot virus, can lay a good foundation for the subsequent development of a kit for detecting the serum of the tomato ringspot virus, and serves the requirements of entry-exit inspection and quarantine work.
Drawings
FIG. 1 shows the antigen sequence and the position of candidate antigenic peptide 7 on the coat protein of tomato ringspot virus.
Detailed Description
The invention is further illustrated by the following examples: