A chicken-derived recombinant full-length monoclonal IgY antibody against human TK1 and its application

Abstract

The present invention discloses a chicken - derived recombinant full - length monoclonal IgY antibody against human thymidine kinase 1 (TK1) (hTK1 - IgY - rmAb) and its application. By an improved phage display method, using the C - terminal 195 - 225 polypeptide of human TK1, a chicken - derived recombinant full - length monoclonal IgY antibody against human TK1 was developed. A super - large library of 3.4×10<supgt;9< / supgt> pfu / mL was rapidly constructed within three weeks, and the positive clone rate was as high as 98%. By using ELISA, immunoblotting and immunohistochemical detection methods, an optimized hTK1 - IgY - rmAb antibody with high specificity and high sensitivity was identified. The present invention improves the stability between batches of the antibody, enables the operation of a new generation of TK1 detection kits based on recombinant IgY antibodies on an automated chemiluminescence analyzer, and can meet the detection requirements for large - scale early - stage tumor risk screening.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody engineering, and particularly relates to a chicken-derived recombinant full-length monoclonal IgY antibody against human TK1 and its application. Background Art

[0002] Thymidine kinase 1 (TK1) is a kinase in the pyrimidine salvage pathway. With the participation of magnesium ions and ATP, it catalyzes the conversion of deoxythymidine (dT) into deoxythymidine monophosphate (dTMP), participates in the synthesis of DNA in the S phase of the cell cycle, and is a key enzyme for cell cycle regulation. The content of TK1 in normal adult cells is extremely low (<2 pmol / L). Only when cells proliferate excessively, the concentration of TK1 will increase and be released into the body fluid, resulting in an increase in the level of serum thymidine kinase 1 (STK1). TK1 is a key enzyme involved in DNA synthesis during the cell cycle and is closely related to the cell proliferation rate. The content of TK1 in the serum of normal healthy individuals without any tumor diseases is extremely low, but in tumor patients with abnormal cell proliferation, TK1 can be easily detected in both serum and tissues. The increase in TK1 is significantly correlated with the proliferation rate of tumor cells in patients with malignant tumors. It is a recognized accurate biomarker for evaluating the proliferation degree of tumor cells and is used for early risk screening of different types of malignant tumors, evaluation of the treatment effects of surgery, radiotherapy and chemotherapy, prediction of the risk of tumor recurrence, etc.

[0003] Currently, there are a series of mouse anti-human TK1 IgG monoclonal antibodies (mAbs) on the market, such as those from AroCell in Sweden (www.arocell.com), and rabbit-derived recombinant monoclonal antibodies against human TK1, such as those from Abcam in the United States (https: / / www.abcam.com). However, in serological TK1 detection, most of these antibodies are only used for scientific research and lack verification of large-scale clinical detection performance in serology. Using these mAbs, the treatment effects of cancer patients can be evaluated through serological TK1 levels. However, due to the poor specificity and sensitivity of these mAbs, they cannot be used for the discovery and risk process assessment of early tumors in health check-ups, nor are they suitable for the prognosis assessment of tumor patients. There is currently no large sample literature data published.

[0004] The commercial enhanced immune dot blot chemiluminescence method (ECL-dotblot) developed by Huarui Tongkang Biotechnology (Shenzhen) Co., Ltd. uses the C-terminal peptide (195-225) of human TK1 to develop chicken anti-human TK1-IgY polyclonal antibodies (TK1-IgY-pAbs), which have been successfully used to detect the concentration of thymidine kinase 1 protein (STK1p) in human serum. Its application in clinical oncology and physical examination screening has exceeded 15 years, verifying that TK1-IgY-pAbs can not only be used as a reliable and accurate target for evaluating the prognosis and outcome of cancer patients, but also for assessing the prognosis, recurrence risk and survival effect of cancer patients. In particular, it can be used for early warning assessment of the risk of tumor progression. Based on the data of 160,086 physical examination subjects in four independent routine health examination centers in China from 2005 to 2016, the high value of STK1p detected by TK1-IgY-pAbs can indicate the risk degree of benign and malignant tumors, that is, low-risk or high-risk groups, before imaging abnormalities occur. It is applicable to different occupations and groups, especially in areas with a high incidence of cancer. It has important value for early tumor risk screening and health management, such as industries exposed to carcinogenic chemicals, etc., to achieve the goal of "early screening, early prevention, early detection and early treatment" of tumors.

[0005] For the previous generation of products, due to the individual differences and living environments of the immunized hens, it is necessary to regularly detect whether each hen produces antibodies with high stability, good specificity and high sensitivity to control the detection performance of antibodies in each production batch. These control works not only require fine preparation processes and experienced technicians, but also consume a long time cycle and high cost investment. Therefore, there is an urgent need to further improve the IgY polyclonal antibody products obtained from the previous generation of traditional immunized hens. Summary of the Invention

[0006] In order to improve the stability between batches of antibodies, the present application provides a chicken-derived anti-human TK1 recombinant full-length monoclonal IgY antibody and its application.

[0007] In a first aspect, the present application provides a chicken-derived anti-human TK1 recombinant IgY single-chain antibody. The single-chain antibody is composed of a heavy chain, a linker peptide, and a light chain. The heavy chain includes a first heavy-chain variable region, a second heavy-chain variable region, and a third heavy-chain variable region. The light chain of the single-chain antibody includes a first light-chain variable region, a second light-chain variable region, and a third light-chain variable region. The amino acid sequence of the first heavy-chain variable region is selected from one of SEQ ID NO: 1 to SEQ ID NO: 3. The amino acid sequence of the second heavy-chain variable region is selected from one of SEQ ID NO: 4 to SEQ ID NO: 6. The amino acid sequence of the third heavy-chain variable region is selected from one of SEQ ID NO: 7 to SEQ ID NO: 9. The amino acid sequence of the first light-chain variable region is selected from one of SEQ ID NO: 10 to SEQ ID NO: 14. The amino acid sequence of the second light-chain variable region is selected from one of SEQ ID NO: 15 to SEQ ID NO: 19. The amino acid sequence of the third light-chain variable region is selected from one of SEQ ID NO: 20 to SEQ ID NO: 23

[0008] Preferably, the variable regions of the same single-chain antibody are composed of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 of the heavy chain and SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20 of the light chain, or composed of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 of the heavy chain and SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21 of the light chain, or composed of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 of the heavy chain and SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 20 of the light chain, or composed of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9 of the heavy chain and SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 22 of the light chain, or composed of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 of the heavy chain and SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 23 of the light chain.

[0009] SEQ ID NO: 1 DRGMG;

[0010] SEQ ID NO: 2 NHGMG;

[0011] SEQ ID NO: 3 RHGMG;

[0012] SEQ ID NO: 4 GVDDSGSSRDYAPAVKGRAT;

[0013] SEQ ID NO: 5 GINSGAGSYTNYGAAVKGRAT;

[0014] SEQ ID NO: 6 SINSGGSYTNYGAAVQGRAT;

[0015] SEQ ID NO: 7 GSVVGFIDA;

[0016] SEQ ID NO: 8 SVYGGSISLITDVIDT;

[0017] SEQ ID NO: 9 DAGCTGGGTCYAYADRIDA;

[0018] SEQ ID NO: 10 SGGGSYAGSYY;

[0019] SEQ ID NO: 11 SGGANYYGSYYYG;

[0020] SEQ ID NO: 12 SGGGRFAGSYYYG

[0021] SEQ ID NO: 13 SGSSGSYG;

[0022] SEQ ID NO: 14 SGSYSNYYG;

[0023] SEQ ID NO: 15 NNNNRP;

[0024] SEQ ID NO: 16 DNTKRP;

[0025] SEQ ID NO: 17 DSTNRP;

[0026] SEQ ID NO: 18 YNGKRP;

[0027] SEQ ID NO: 19 ENNNRP;

[0028] SEQ ID NO: 20 GSIDSSYVGI;

[0029] SEQ ID NO: 21 GSYDSSAGYAG;

[0030] SEQ ID NO: 22 GSRDSSYDGI;

[0031] SEQ ID NO: 23 GGYDDSTDSGI;

[0032] In a second aspect, the present application provides a chicken-derived recombinant full-length monoclonal IgY antibody against human TK1, which is characterized by: comprising the above heavy chain and light chain, and there are 5 monoclonal antibodies in total, with the amino acid sequences of SEQ ID NO: 24 to SEQ ID NO: 28 respectively.

[0033] SEQ ID NO: 24

[0034] TK1-hen-1 AVTLDESGGGLQTPGGALSLVCKASGFTFSDRGMGWMRQAPGKGLEFVAGVDDSGSSRDY 60

[0035] TK1-hen-1 APAVKGRATISRDNGQSTLSLQLSDLRAEDTGTYYCTRGSVVGFIDAWGHGTEVIVSSG119

[0036] TK1-hen-1 GGGSGGGGSGGGGSQAALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKSP175

[0037] TK1-hen-1 GSAPVTVIYNNNNRPSGIPSRFSGSLSGSTNTLTITGVQAEDEAIYYCGSIDSSYVGIFG 235

[0038] TK1-hen-1 AGTALTVL 243.

[0039] SEQ ID NO: 25

[0040] K1-hen-2 AVTLDESGGGLQTPGGGLSLVCKASGFTFSNHGMGWVRQAPGKGLEWVAGINSGAGSYTN60

[0041] TK1-hen-2 YGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYFCARSVYGGSISLITDVIDTWGHG119

[0042] TK1-hen-2 TEVTVSSGGGGSGGGGSGGGGSQAALTQPASVSANLGETVKITCSGGANYYGSYYY175

[0043] TK1-hen-2 GWYQQKSPGSAPVTVINDNTKRPSNIPSRFSGSTSGSTGTLTITGVQADDEAVYFCGSYD 235

[0044] TK1-hen-2 SSAGYAGIFGAGTTLTVL253。

[0045] SEQ ID NO: 26

[0046] TK1-hen-3 AVTLDESGGGLQTPGGTVSLVCKASGFTFSDRGMGWMRQAPGKGLEFVAGVDDSGSSRDY 60

[0047] TK1-hen-3 APAVKGRATISRDNGQDILRLQLNNLRAEDTATYYCTRGSVVGFIDAWGHGTEVIVSSG120

[0048] TK1-hen-3 GGGSGGGGSGGGGSQAALTQPASVSASPGETVKITCSGGGRFAGSYYYGWYQQKSP175

[0049] TK1-hen-3 GSAPVTVIYDSTNRPSNIPSRFSGSLSGSTTTLTITGVQADDEAVYFCGSIDSSYVGIFG 235

[0050] TK1-hen-3 AGTTLTVL 243。

[0051] SEQ ID NO: 27

[0052] TK1-hen-4 AVTLDESGGGLQTPGGALSLVCKASGFSFSRHGMGWVRQAPGKGLEWVASINSGGSYTNY 60

[0053] TK1-hen-4 GAAVQGRATIWRDNGQSTVRLQLNNLRAEDTATYYCARDAGCTGGGTCYAYADRIDAWG119

[0054] TK1-hen-4 HGTEVIVSSGGGGSGGGGSGGGGSQAALTLPSSVSVNPGETVKITCSGSSGSYGWY175

[0055] TK1-hen-4 QQKSPGSAPVTVIYYNGKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSRDSS235

[0056] TK1-hen-4 YDGIFGAGTTLTVL 248。

[0057] SEQ ID NO: 28

[0058] TK1-hen-5 AVTLDESGGGLQTPGGGLSLVCKASGFTFSNHGMGWVRQAPGKGLEWVAGINSGAGSYTN 60

[0059] TK1-hen-5 YGAAVKGRATISRDNGQSTVRLQLNNLRAEDTATYYCARSVYGGSISLITDVIDTWGHG119

[0060] TK1-hen-5 TEVIVSSGGGGSGGGGSGGGGSQAALTQPSSVSANPGETVKITCSGSYSNYYGWYQ175

[0061] TK1-hen-5 QKSPGSAPVTVIYENNNRPSDIPSRFSGSKSGSTNTLTITGVQVEDEAVYFCGGYDDSTD 235

[0062] TK1-hen-5 SGIFGAGTTLTVL 248。

[0063] Preferably, the amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO: 28.

[0064] In a third aspect, the present application provides a recombinant vector, the recombinant vector contains nucleic acid encoding the above amino acids, and the recombinant vector is constructed with the PTT5 plasmid.

[0065] In a fourth aspect, the present application provides a cell containing the recombinant vector, the cell contains the above recombinant vector, and a stable cell line is constructed using Chinese hamster ovary (CHO) cells.

[0066] In a fifth aspect, the present application provides a test card, a test strip or a test kit, the test card, the test strip or the test kit contains any one of the above single-chain antibodies or monoclonal antibodies.

[0067] In a sixth aspect, the present application provides a drug containing any one of the above antibodies.

[0068] Seventh aspect, the present application provides a method for screening any of the above-mentioned antibodies, comprising the following steps:

[0069] Any of the above antibodies can rapidly construct a large-capacity phage antibody display library within three weeks through step 1) separating mRNA from hen spleen cells, step 2) PCR reverse transcription, step 3) PCR overlap to obtain the full-length scFv gene, step 4) splicing and concentrating scFv, step 5) electrotransformation in highly active bacterial competent cells, step 6) phage antibody packaging, and step 7) screening.

[0070] Preferably, within 15 minutes after the hen is sacrificed in step 1), spleen cells are quickly taken, homogenized with TriZol solution, and total RNA is separated with chloroform and isopropanol;

[0071] In step 2), oligodT is used as a primer to perform reverse transcription reaction to prepare cDNA for subsequent amplification of antibody genes;

[0072] In step 3), degenerate primers are used to amplify the genes of the heavy chain variable region and the light chain variable region, and assembled into scFv gene by overlap PCR (polymerase chain reaction);

[0073] In step 4), the PCR product is separated and digested with Sfi I and Not I before being ligated to the phagemid vector pSCD-1 (as shown in the appendix Figure 1 );

[0074] In step 5), the ligation mixture is desalted and electrotransformed into TG1 competent cells;

[0075] In step 6), the helper phage M13KO7 is added to the phagemid vector shown in the appendix Figure 1 After packaging, the amplified phage is precipitated with PEG 6000 and titrated for further screening;

[0076] In step 7), the phage library is added to a screening plate containing the target TK1. After multiple washings and incubations, the bound phage is digested and eluted, amplified, and then subjected to multiple rounds of screening. Single colonies are taken out and inoculated for culture. When the absorbance value at 600 nm reaches 0.4 - 0.6, the helper phage is added for culture at a multiplicity of infection (MOI) between 10 - 20, and the phage is detected by enzyme-linked immunosorbent assay (ELISA) and DNA sequencing. According to the DNA sequencing results, all unique single-chain antibodies are recombined. Based on the backbone of the IgY full-length antibody heavy chain and light chain sequences, the full-length antibody sequence is constructed through consecutive PCR and cloning steps, and then loaded into the eukaryotic expression vector PTT5 to obtain the PTT5-TK1-IgY-H and PTT5-TK1-IgY-L expression vector pairs.

[0077] In an eighth aspect, the present application provides a detection method using a chicken-derived anti-human TK1 recombinant IgY antibody containing any one of the above single-chain antibodies or monoclonal antibodies. By applying any one of the above antibodies, the concentration of TK1 in body fluid samples (including blood, urine, saliva, cerebrospinal fluid, cavity effusion, etc.) is manually or automatically detected by enhanced chemiluminescence (ECL) dot blotting or chemiluminescence immunoassay.

[0078] In the previous generation of products, the inventors mastered how to preferably obtain TK1-IgY polyclonal antibodies with strong specificity and high sensitivity from immunized hens. Drawing on the existing improved recombinant antibody development technology, its preparation process is easier to standardize, and at the same time, it can reduce the consumption of time, manpower, and cost, which is particularly important and necessary for the development of TK1-IgY monoclonal antibodies. This will enable the mass production of antibodies with strong specificity, high sensitivity, and high stability, ensuring the large-scale detection needs in population physical examination screening and clinical oncology.

[0079] Based on the existing TK1-IgY polyclonal antibody technology, the inventors developed recombinant chicken IgY-TK1-mAb. Since the recombinant monoclonal IgY antibody is an antibody derived from a single B cell lineage, this lineage has undergone repeated somatic hypermutation and clonal selection, and is usually an IgY isotype that recognizes a single unique epitope. The phage display technology can now be used to successfully and rapidly develop recombinant anti-TK1 monoclonal IgY antibodies. Compared with the corresponding natural polyclonal IgY antibodies, the recombinant anti-TK1 monoclonal IgY antibodies have stronger specificity, higher affinity, and stability. The recombinant antibodies also have advantages such as editability, and can be modified in terms of antibody stability, affinity, antibody modification, etc. according to actual needs to meet different new antibody application requirements. The inventors' team successfully developed a chicken-derived anti-human TK1 recombinant full-length monoclonal IgY antibody for the first time, named "human TK1 recombinant-IgY-monoclonal antibody". The new generation of human TK1 recombinant-IgY-monoclonal antibody will provide an innovative prospect for the research and application of early tumor risk screening and tumor medicine.

[0080] Cancer is a chronic proliferative disease of tumor cells, mainly caused by gene mutations in certain enzymes and proteins related to cell growth regulation, resulting in uncontrolled and infinite proliferation of cells, thus leading to the occurrence of malignant tumors. The non-invasive serological TK1 detection method provides information on the early risk process of tumors that cannot be achieved by imaging. Therefore, the TK1 serological biomarker is of great value for predicting the risk of malignant tumor progression. The TK1 serological biomarker, combined with imaging and other applicable tools, will enable long-term follow-up to detect precancerous lesions or early risk signs of tumors that cannot be detected by imaging. The inventors designed a specific TK1-31 peptide antigen, successfully prepared a chicken anti-human TK1-IgY antibody, developed and established a commercial high-sensitivity immunochemiluminescent dot blot (ECL dot blot) detection system to measure the concentration of TK1 protein (STK1p) in serum, thus replacing the detection method for TK1 activity (STKa) in serum. Based on the Meta study of 35,365 large-scale serum samples from the physical examination population, the inventors set STK1p = 2.0 pM as a scientifically reasonable "risk threshold" to effectively evaluate the abnormal cell growth rate in the routine population screening. In this Meta study, using the internationally recognized receiver operating characteristic curve (ROC) analysis method, preoperative patients (n = 720) with 10 different types of malignant tumors and people without malignant tumors / without tumor-related diseases (n = 4,103) were analyzed, and the area under the curve was 0.96. When the best specificity was 0.997, the sensitivity was 0.80, and the maximum likelihood value (+) was 236.5, the risk threshold was 2.0 pM. Using the risk threshold of 2.0 pM, the effectiveness of the TK1 kit assembled by the inventors' enhanced chemiluminescent dot blot immune system was verified in the routine population physical examination screening. In this Meta study, it was found that the incidence of new malignant tumors in the high-risk group of STK1p (STK1p > 2.0 pM) within 6 years was 3-5 times higher than the new incidence rate of Chinese tumor statistics (0.2% - 0.3%). Compared with the low-risk group of STK1p (≤2.0 pM), the risk of new malignant tumors in the high-risk group of STK1p was even higher (44 times) within 11 years. The inventors' research confirmed that STK1p is more suitable for the efficacy monitoring of various tumors, the evaluation of recurrence and survival rate of cancer patients, especially suitable for the early detection of malignant tumors and the prediction of the risk process of tumor development, and is an optimal biomarker for predicting malignant tumors and predicting the risk of tumor development.

[0081] This application uses the C-terminal 195-225 polypeptide of human TK1 to develop a chicken anti-human TK1 full-length-IgY monoclonal antibody. Through the improved phage display method, the positive clone rate is as high as 98%, and 3.4×10 can be rapidly constructed within 3 weeks 9A pfu / mL ultra-large library can completely eliminate environmental phage contamination. After successfully obtaining single-chain antibodies that meet the requirements of the inventors, the variable region sequences of the heavy and light chains of the antibody are obtained. After analyzing the obtained antibody fragments, the full-length IgY antibody is constructed, and then the full-length antibody is expressed through a eukaryotic expression system and purified. Currently, ELISA titer determination, immunoblotting, and immunohistochemical detection of this antibody have verified that this antibody is an antibody with high specificity and sensitivity. More importantly, serological identification indicates that the results of using the two serum detection kits assembled by the inventors show that the coincidence rate of the detection results of the two kits is as high as r = 0.857. In the future, the fully automated chemiluminescence analyzer will replace the past semi-automated enhanced chemiluminescence immunoassay system, enabling the new generation of TK1-IgY recombinant antibody kits to run on the fully automated chemiluminescence analyzer to meet the detection needs of large-scale early tumor risk screening. Currently, the successful construction of the TK1-IgY-rmAb antibody using phage display library technology and its operation on the fully automated chemiluminescence analyzer have not been reported, which is the first in the world.

[0082] The inventors successfully obtained the TK1-IgY-rmAb antibody using phage display library technology. The currently constructed stable cell line ultimately aims to prepare a large number of TK1-IgY-rmAb antibodies with high specificity, high sensitivity, and stability, and assemble detection kits for serum detection for early tumor risk screening in routine population physical examinations. In the future, the TK1-IgY-rmAb kit will show great potential in early tumor risk screening in routine population physical examinations, and can be used to predict pre-cancerous diseases and (or) tumor-related risk diseases, as well as evaluate the risk prognosis possibility of developing into malignant tumors in the future, and give patients timely early intervention treatment. And this new generation of TK1-IgY-rmAb kit is also applicable to clinical oncology, including follow-up of tumor patients and monitoring of treatment effects, prediction and evaluation of recurrence and survival.

[0083] Phage display technology has become an important method for preparing monoclonal antibodies from immune and non-immune sources. This method is simple, fast, time-consuming, and can quickly screen and obtain monoclonal antibodies without being restricted by the conventional hybridoma method. It is currently the most mature and widely used gene engineering antibody screening technology. Phage display technology has the advantages of large screening capacity, high efficiency, easy operation at the gene level, and easy large-scale production. Its library capacity is as high as 10 9 -10 12pfu / mL, it can select millions to hundreds of millions of molecules, and each round of panning can enrich clones with stronger antigen specificity; making the screening of specific antibodies easier. This method can modify antibodies according to different purposes, broadening the application scope of antibodies. At present, phage display technology has made significant achievements in antibody applications, but most studies obtain antibody libraries from immunized mammals. Research shows that some highly conserved mammalian antigens usually have the problem of low immunogenicity during the immunization of mammals. Brief Description of the Drawings

[0084] Figure 1 It is a schematic diagram of the phagemid pSCD-1.

[0085] Figure 2 It is the construction of a phage antibody library, DNA (2000, 1000, 750, 500, 250, and 100 bp). Among them, A is the isolation of RNA, B is the second-round PCR of the antibody light chain, C is the second-round PCR of the antibody heavy chain, D is the splicing of the scFv gene by overlap PCR, and E is the detection of library diversity.

[0086] Figure 3 It is the recombinant full-length protein TK1-IgY-rmAb 5 # Eukaryotic expression, detecting the antibody titer by the Elisa titer method (Elisa titer) (the antibody concentration is serially diluted from 1000 to 7.8 ng / mL), Figure 3 The denaturing SDS-PAGE gel immunoelectrophoresis diagram is shown in the upper right corner.

[0087] Figure 4 A shows the TK1 immunohistochemistry (HIC) staining of normal tonsil tissue and TK1 staining in tissues of patients with poorly differentiated ovarian cancer.

[0088] Figure 4 B is TK1-IgY-rmAb 5 # Results for ECL dot blotting and automated chemiluminescence methods.

[0089] Figure 5 Results of hTK1-IgY-rmAb and hTK1-IgY-pAb for ECL dot blotting. Detailed Implementation Modes

[0090] Example 1

[0091] This example discloses the preparation process of a chicken-derived recombinant full-length monoclonal IgY antibody against human TK1

[0092] 1. Construction of the TK1-IgY-scFv phage library

[0093] Step 1), isolating mRNA from hen spleen cells: Immunize hens with a polymer of artificially synthesized human TK1-31 peptide (GQPAGPDNKENCPVPGKPGEAVAARKLFAPQ) and KLH. Collect eggs and isolate the crude TK1-IgY-pAb polyclonal antibody from egg yolks, and then obtain the purified TK1-IgY-pAb antibody through affinity purification method. Identify the specificity and sensitivity of the antibody by immunoblotting, immunohistochemistry and serology. Select hens that produce highly specific and sensitive TK1-IgY-Abs for library construction. Sacrifice the hens and quickly collect spleen cells within 15 min, and homogenize them with TriZol solution (Invitrogen).

[0094] Step 2) PCR reverse transcription, separating total RNA with chloroform and isopropanol, and then using oligodT as primer to perform reverse transcription reaction to prepare cDNA for subsequent amplification of antibody genes.

[0095] Step 3), overlapping PCR to obtain the full-length scFv gene: The cDNA was prepared using the 1 st Strand cDNA Synthesis Kit from TaKaRa™ Prime Script. Degenerate primers were used to amplify the VH and VL genes. They were assembled into the scFv gene through overlapping PCR (Polymerase Chain Reaction).

[0096] Step 4), splicing and concentrating scFv: Isolate the PCR product and cut it with Sfi I and Not I before ligating it to the phagemid vector pSCD-1 ( Figure 1 ).

[0097] Step 5), performing electrotransformation in highly active bacterial competent cells: Desalt the ligation mixture and electrotransform it into TG1 competent cells.

[0098] Step 6), packaging the phage antibody library: Add the helper phage M13KO7 to the phagemid vector. The packaged phages are precipitated with PEG 6000 and subjected to routine titration to prepare for further screening process. The construction of the library is as Figure 2 shown. Complete the rapid construction of a large-capacity phage antibody display library within 3 weeks, see attachment Figure 2 . Finally, obtain a phage library with antibody diversity. The library size is 3.4×10 9 pfu / ml (pfu, plaque forming unit), for subsequent screening. The calculation of the antibody library capacity is shown in Table 1. Randomly select 50 monoclonal clones for colony PCR identification, and the amplification band sizes of 49 clones are correct, and the positive clone rate is as high as 98%.

[0099] Table 1. Method for calculating storage capacity

[0100]

[0101] 2. Screen qualified positive clones.

[0102] Add the phage library to a well-sealed 96-well plate coated with the target TK1 and incubate at 37 °C for 1 h. After washing 9 times with PBST, the bound phages are eluted by trypsin digestion. Finally, with the help of M13KO7 helper phage, the eluate is amplified and titrated for the next round of screening. A total of three rounds of screening are performed. The content of Tween 20 in PBST is increased successively in each round (i.e., 0.1% Tween 20-PBS in the first round, 0.2% Tween 20-PBS in the second round, and 0.3% Tween 20-PBS in the third round), and the coating concentration of TK1 antigen is reduced from the initial concentration of 10 μg / mL in each round to 2 μg / mL.

[0103] Calculate the enrichment efficiency of each round. The calculation results show that the ratio of the output phages to the input phages increases steadily after each round of panning (Table 2). Compared with the first round of screening, the phage recovery rate increases by about 923 times after the third round of screening, demonstrating the effective enrichment of specific antibodies. The panned library is used for subsequent selection of high-affinity antibodies.

[0104] After the end of the third round of panning, pick single colonies (without amplification) from the final eluate and inoculate them into a 96-well plate containing 100 μL of 2YT-AG (2YT medium, 100 μg / mL ampicillin, 0.1% glucose), and culture overnight at 37 °C / 220 rpm. The next day, transfer 20 μL of the overnight culture to a new 96-well plate containing 80 μL of 2YT-A (2YT medium, 100 μg / mL ampicillin). When OD600 reaches about 0.5, add M13KO7 at an MOI of 10 - 20 and culture with shaking at 37 °C / 180 rpm for 30 min. Subsequently, add 100 μL of 2YT-AK (2YT medium, 100 μg / mL ampicillin, 50 μg / mL kanamycin) to each well and culture overnight at 37 °C / 220 rpm. Finally, transfer 100 μL of the supernatant from each well to a assay plate coated with the TK1 target for Phage Elisa detection.

[0105] Table 2. Process of phage library panning

[0106] Washing times Input (pfu) Output (pfu) Enrichment rate 1 <![CDATA[5.0×10 12 > <![CDATA[9.6×10 3 > <![CDATA[1.92×10 -9 > 2 <![CDATA[5.24×10 12 > <![CDATA[7.44×10 4 > <![CDATA[1.42×10 -8 > 3 <![CDATA[5.28×10 12 > <![CDATA[9.36×10 6 > <![CDATA[1.77×10 -6 >

[0107] To identify the quality of the antibody library, the antibody library needs to be sequenced: The positive clones selected by Phage ELISA are inoculated into LB-A (LB medium, 100 μg / mL ampicillin) and cultured overnight at 37°C / 220 rpm. The plasmids of each clone are isolated using a commercial kit. After DNA sequencing, the sequence data is analyzed using Geneious software.

[0108] A total of 49 samples were sequenced, 44 were successfully sequenced, and the remaining 5 failed. Among the 44 clones, 35 were correct clones, and the remaining 9 had frameshift mutations. The 35 clones were all different and were unique sequences.

[0109] The affinity was detected using the limiting dilution method (0.125 μg - 10 μg / well of TK1 antigen). The results showed that 5 positive clones had high affinity. The sequence of the variable region of the protein gene sequences corresponding to these 5 clones is as follows:

[0110] Gene sequence one:

[0111] GCTGTAACGCTTGATGAATCTGGTGGGGGTTTACAAACACCGGGAGGAGCATTGTCGCTCGTTTGTAAGGCCAGCGGGTTCACATTCAGCGACAGGGGTATGGGATGGATGAGGCAAGCGCCCGGAAAAGGGCTAGAGTTTGTTGCGGGTGTAGATGATTCGGGTAGCAGTAGAGATTATGCCCCCGCAGTGAAAGGTAGGGCTACAATATCTAGGGATAACGGCCAGAGCACGCTAAGCCTCCAATTAAGTGATCTTCGCGCTGAAGACACGGGCACGTATTATTGCACACGGGGTTCCGTAGTAGGGTTCATAGACGCATGGGGTCACGGGACGGAAGTCATAGTATCATCAGGGGGAGGCGGATCCGGTGGCGGGGGATCCGGAGGTGGTGGTTCCCAGGCAGCGTTGACACAGCCGGCGTCAGTCAGTGCGAACCCAGGGGAAACGGTGAAAATTACGTGCTCCGGTGGAGGCTCTTATGCAGGGTCTTATTACTATGGTTGGTATCAACAAAAATCTCCTGGCAGTGCCCCAGTCACTGTCATTTATAATAACAACAACCGACCTAGTGGTATTCCGTCGCGTTTTAGCGGTTCCCTTTCGGGCTCCACGAATACCCTCACGATTACGGGCGTCCAAGCTGAAGACGAAGCGATCTACTACTGCGGATCTATCGACTCTTCCTACGTAGGTATATTTGGTGCCGGCACCGCTTTGACTGTATTA

[0112] Heavy chain variable region: DRGMGGVDDSGSSRDYAPAVKGRATGSVVGF-----IDA.

[0113] Light chain variable region: SGGGSYAGSYYNNNNRPGSIDSS-YVGI.

[0114] Gene sequence two:

[0115] GCGGTAACATTAGACGAGTCAGGCGGGGGACTTCAAACGCCGGGAGGGGGCTTGTCTCTAGTATGCAAAGCGTCTGGATTTACCTTCAGCAATCATGGCATGGGGTGGGTGCGCCAAGCTCCTGGAAAAGGCTTGGAGTGGGTTGCAGGAATCAACAGCGGGGCAGGCTCTTACACAAACTACGGTGCAGCAGTAAAAGGTAGGGCAACCATCAGCCGAGACAACGGACAAAGCACAGTCAGGTTGCAGCTCAACAATTTAAGGGCAGAAGACACTGGCACATACTTCTGCGCACGGTCTGTCTACGGCGGTAGCATATCTCTCATAACGGATGTAATCGACACGTGGGGCCATGGGACGGAAGTGACAGTTTCATCCGGTGGTGGAGGGAGTGGTGGTGGAGGCTCCGGTGGTGGAGGGTCACAAGCAGCCCTGACACAACCGGCAAGCGTCTCAGCTAACTTGGGAGAAACAGTTAAAATCACCTGCTCTGGTGGTGCTAATTACTATGGCAGCTATTATTACGGATGGTACCAACAAAAAAGTCCAGGGTCAGCACCTGTCACAGTCATCAATGACAACACGAAACGCCCATCTAACATACCGTCCAGGTTTTCTGGCAGCACAAGCGGAAGTACAGGCACTCTCACAATTACGGGAGTGCAGGCCGACGATGAAGCTGTCTATTTCTGTGGCTCTTACGACTCTAGCGCTGGCTACGCAGGTATATTTGGAGCAGGTACAACTTTAACAGTATTA

[0116] Heavy chain variable region: NHGMGGINSGAGSYTNYGAAVKGRATSVYGGSISLITDVIDT.

[0117] Light chain variable region: SGGANYYGSYYYGDNTKRPGSYDSSAGYAG.

[0118] Gene sequence three:

[0119] GCAGTAACATTGGATGAATCCGGTGGGGGTTTGCAAACTCCTGGTGGGACAGTCTCACTCGTGTGTAAGGCGTCAGGTTTCACTTTTTCCGATCGGGGTATGGGATGGATGCGGCAAGCGCCGGGCAAAGGACTTGAATTCGTAGCCGGAGTAGATGACTCTGGGTCCAGTCGTGACTACGCGCCCGCTGTCAAAGGTCGGGCGACGATAAGTCGTGACAATGGTCAAGACATATTACGGCTCCAGCTAAACAATCTACGTGCTGAAGATACAGCAACATATTATTGTACGAGAGGCTCAGTGGTAGGTTTCATAGATGCATGGGGACACGGCACGGAGGTTATAGTTTCCTCCGGTGGGGGAGGCTCTGGAGGCGGCGGTTCTGGCGGCGGAGGGTCACAAGCGGCGTTAACCCAACCCGCGTCAGTCTCAGCTTCCCCCGGGGAAACGGTCAAGATCACCTGTTCCGGGGGGGGGCGTTTTGCTGGATCTTATTACTATGGATGGTATCAACAGAAATCCCCCGGTAGTGCCCCGGTGACCGTTATTTACGACTCGACAAATCGGCCTTCTAATATACCGAGTCGTTTCTCTGGTAGCTTGTCTGGCTCAACAACAACGCTAACGATAACCGGTGTTCAGGCAGATGACGAGGCTGTGTATTTCTGCGGCTCTATCGACAGCAGCTATGTAGGGATATTCGGAGCTGGCACGACTCTTACCGTGTTA

[0120] Heavy chain variable region: DRGMGGVDDSGSSRDYAPAVKGRATGS------VVGFIDA.

[0121] Light chain variable region: SGGGRFAGSYYYGDSTNRPGSIDSS-YVGI.

[0122] Gene sequence four:

[0123] GCTGTCACTCTAGACGAGTCAGGAGGCGGACTCCAGACACCTGGGGGGGCCCTAAGCTTGGTTTGTAAAGCATCAGGGTTTTCCTTTTCACGACATGGAATGGGATGGGTCCGGCAAGCCCCGGGAAAAGGACTGGAGTGGGTTGCGAGTATTAATTCAGGGGGATCGTATACGAACTATGGCGCAGCCGTGCAGGGAAGAGCGACTATATGGCGTGATAATGGTCAATCAACGGTAAGGCTACAATTGAATAACCTCCGGGCCGAGGATACGGCAACCTATTATTGTGCGCGAGATGCGGGATGCACTGGTGGCGGCACATGTTATGCATACGCGGACCGTATTGATGCGTGGGGACATGGGACGGAAGTTATTGTATCTAGTGGGGGCGGAGGCTCTGGGGGGGGGGGGTCGGGCGGTGGGGGCTCCCAGGCAGCGTTAACGCTCCCGAGCTCTGTGTCCGTGAATCCAGGAGAAACTGTTAAAATCACATGTTCAGGCTCTAGCGGCAGCTACGGCTGGTATCAACAAAAATCCCCCGGTTCCGCTCCAGTCACCGTTATTTACTACAATGGGAAGAGGCCGTCGGATATACCGTCTCGCTTCTCCGGTTCTAAATCCGGCTCAACGGGGACACTCACCATAACTGGCGTACAAGCTGAGGACGAGGCGGTGTACTTTTGCGGTAGTCGTGATAGCTCATACGACGGCATATTTGGAGCTGGTACAACACTGACGGTGCTT

[0124] Heavy chain variable region: RHGMGSINSGGSYTNYGAAVQGRATDAGCTGGGTCYAYADRIDA.

[0125] Light chain variable region: SGSS---GS--YGYNGKRPGSRDSSYDGI.

[0126] Gene sequence five:

[0127] GCGGTTACACTAGATGAGAGCGGAGGTGGTCTACAAACGCCTGGTGGGGGTTTATCCCTCGTATGTAAAGCGTCTGGCTTCACATTTTCCAACCACGGGATGGGATGGGTACGCCAGGCACCCGGTAAAGGTCTCGAATGGGTGGCTGGAATAAATAGCGGAGCAGGATCTTATACCAACTACGGCGCAGCCGTTAAGGGGCGTGCAACTATCTCCCGCGACAATGGACAGTCCACCGTTCGACTCCAACTGAATAACCTTCGGGCTGAGGACACTGCTACTTATTACTGCGCCCGTTCGGTTTATGGTGGGAGTATTTCGTTAATTACAGATGTTATTGACACATGGGGCCACGGCACTGAGGTCATCGTCTCTTCTGGTGGGGGCGGCTCAGGAGGAGGCGGGTCTGGAGGAGGCGGTTCGCAGGCGGCCCTTACGCAACCATCAAGTGTCAGTGCAAATCCGGGAGAAACTGTTAAGATAACCTGTTCCGGATCTTACAGCAATTATTATGGCTGGTATCAGCAAAAATCTCCGGGGAGTGCTCCTGTTACTGTGATATACGAAAACAACAACAGGCCCAGCGATATTCCTTCACGTTTTTCGGGATCTAAATCTGGTTCTACTAACACTTTGACAATTACTGGTGTCCAGGTAGAGGACGAAGCCGTGTACTTTTGCGGCGGTTACGATGACTCTACGGACAGCGGAATATTCGGCGCAGGCACCACGCTGACCGTACTT

[0128] Heavy chain variable region: NHGMGGINSGAGSYTNYGAAVKGRATSVYGGSISLITDVIDT.

[0129] Light chain variable region: SG—SYSN--YYGENNNRPGGYDDSTDSGI.

[0130] The recombinant full-length antibody protein is expressed eukaryotically.

[0131] The unique sequences of the above 5 scFv positive clones were screened and obtained, and 5 full-length IgY antibodies were constructed, SEQ ID NO: 24-28, which were named TK1-hen-1, 2, 3, 4, and 5 in sequence. First, they were transfected into 293T suspension cells for expression, and the supernatant after 6 days of culture was collected for Elisa detection.

[0132] SEQ ID NO: 24

[0133] TK1-hen-1 AVTLDESGGGLQTPGGALSLVCKASGFTFSDRGMGWMRQAPGKGLEFVAGVDDSGSSRDY 60

[0134] TK1-hen-1 APAVKGRATISRDNGQSTLSLQLSDLRAEDTGTYYCTRGSVVGFIDAWGHGTEVIVSSG119

[0135] TK1-hen-1 GGGSGGGGSGGGGSQAALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKSP175

[0136] TK1-hen-1 GSAPVTVIYNNNNRPSGIPSRFSGSLSGSTNTLTITGVQAEDEAIYYCGSIDSSYVGIFG235

[0137] TK1-hen-1 AGTALTVL 243。

[0138] SEQ ID NO: 25

[0139] TK1-hen-2 AVTLDESGGGLQTPGGGLSLVCKASGFTFSNHGMGWVRQAPGKGLEWVAGINSGAGSYTN60

[0140] TK1-hen-2 YGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYFCARSVYGGSISLITDVIDTWGHG119

[0141] TK1-hen-2 TEVTVSSGGGGSGGGGSGGGGSQAALTQPASVSANLGETVKITCSGGANYYGSYYY175

[0142] TK1-hen-2 GWYQQKSPGSAPVTVINDNTKRPSNIPSRFSGSTSGSTGTLTITGVQADDEAVYFCGSYD235

[0143] TK1-hen-2 SSAGYAGIFGAGTTLTVL 253。

[0144] SEQ ID NO: 26

[0145] TK1-hen-3 AVTLDESGGGLQTPGGTVSLVCKASGFTFSDRGMGWMRQAPGKGLEFVAGVDDSGSSRDY 60

[0146] TK1-hen-3 APAVKGRATISRDNGQDILRLQLNNLRAEDTATYYCTRGSVVGFIDAWGHGTEVIVSSG120

[0147] TK1-hen-3 GGGSGGGGSGGGGSQAALTQPASVSASPGETVKITCSGGGRFAGSYYYGWYQQKSP175

[0148] TK1-hen-3GSAPVTVIYDSTNRPSNIPSRFSGSLSGSTTTLTITGVQADDEAVYFCGSIDSSYVGIFG235

[0149] TK1-hen-3 FGAGTTLTVL 245。

[0150] SEQ ID NO: 27

[0151] TK1-hen-4 AVTLDESGGGLQTPGGALSLVCKASGFSFSRHGMGWVRQAPGKGLEWVASINSGGSYTNY 60

[0152] TK1-hen-4GAAVQGRATIWRDNGQSTVRLQLNNLRAEDTATYYCARDAGCTGGGTCYAYADRIDAWG119

[0153] TK1-hen-4 HGTEVIVSSGGGGSGGGGSGGGGSQAALTLPSSVSVNPGETVKITCSGSSGSYGWY175

[0154] TK1-hen-4QQKSPGSAPVTVIYYNGKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSRDSS235

[0155] TK1-hen-4 YDGIFGAGTTLTVL 248。

[0156] SEQ ID NO: 28

[0157] TK1-hen-5 AVTLDESGGGLQTPGGGLSLVCKASGFTFSNHGMGWVRQAPGKGLEWVAGINSGAGSYTN 60

[0158] TK1-hen-5 YGAAVKGRATISRDNGQSTVRLQLNNLRAEDTATYYCARSVYGGSISLITDVIDTWGHG119

[0159] TK1-hen-5 TEVIVSSGGGGSGGGGSGGGGSQAALTQPSSVSANPGETVKITCSGSYSNYYGWYQ175

[0160] TK1-hen-5 QKSPGSAPVTVIYENNNRPSDIPSRFSGSKSGSTNTLTITGVQVEDEAVYFCGGYDDSTD235

[0161] TK1-hen-5 SGIFGAGTTLTVL 248。

[0162] Detect with 1 µg / mL TK1 target-coated antigen; select 5 antibodies with high affinity and binding ability from 5 antibodies # and name them human TK1-IgY-rmAb 5 # . Subsequently, verify the sensitivity and specificity of the antibody by immunoblotting, immunohistochemistry and serological detection ( Figure 3 and Figure 4 ).

[0163] Figure 3 It is the ELISA titer method to detect TK1-IgY-rmAb 5 #: The titer of the purified antibody was detected using 1 μg / mL TK1 target as the coated antigen. The purified antibody was serially diluted 2-fold to 7.8 ng / mL. It was incubated at 37 °C for 1 h, then washed three times with PBST (0.1% Tween, PBS), after which a secondary antibody conjugated with HRP was added, washed five times with PBST, and finally TMB was added for color development. Detection was performed at OD450 after termination with 1 M H2SO4. The repeated detection results showed ( Figure 3 ) that the antibody had high sensitivity.

[0164] Denaturing SDS-PAGE gel immunoelectrophoresis (Western blot) was performed using a TK1-negative cell line (143BTK1- / -) and a TK1-positive cell line (HT29). 50 ng of cell lysate was loaded respectively, and the usage amount of TK1-IgY-rmAb 5 # was 0.05 μg / mL. The detection results showed that there was an obvious TK1 monomer (about 25 kD) in the TK1-positive cell lysate, which was a specific band of TK1 expression, but there was no specific band of TK1 monomer in the TK1-negative cell lysate, proving that the antibody had good specificity.

[0165] # TK1 tissue immunohistochemistry and TK1 serological identification.

[0166] Immunohistochemical (TK1) staining was performed using a patient with poorly differentiated ovarian cancer (pathological grade 3) and normal tonsil tissue. The normal tonsil tissue contained two different regions, including the proliferative region and the non-proliferative region. It was usually used as a good standard detection method to evaluate whether the antibody could evaluate the cell proliferation rate. Therefore, we used this tissue to evaluate the specificity and sensitivity of TK1-IgY-rmAb 5 # . Figure 4 The results showed that TK1 staining was only observed in the cells of the proliferative region of the tonsil. The characteristics of the staining were mainly varying degrees of brown-yellow staining in the cytoplasm, but no staining was observed in the cytoplasm of tonsil cells in the non-proliferative region. In addition, in patients with poorly differentiated ovarian cancer (pathological grade 3), the characteristics of TK1 staining were mainly varying degrees of brown-yellow staining in the cytoplasm. It was proved that TK1-IgY-rmAb 5 # had high specificity and sensitivity, the same as the IgY-pAb and TK1-IgG monoclonal antibodies we identified in the past.

[0167] The concentrations of STK1p in 292 serum samples were detected using two methods, ECL dot blot and an automatic chemiluminescence analyzer, and then comparative analysis was performed. The analysis results showed ( Figure 4B), the coincidence rate r of the two methods is as high as 0.857 (exceeding 0.75), indicating that the coincidence rate of the two detection methods is good, and the fully automated chemiluminescence analyzer will be more conducive to large-scale early tumor risk screening.

[0168] Example 2

[0169] This example provides a method for preparing a recombinant vector containing a chicken-derived anti-human TK1 recombinant full-length monoclonal IgY antibody, which includes the following steps:

[0170] Step 1), design and synthesis of IgY heavy chain and light chain gene fragments: Integrate the DNA fragment sequences corresponding to the heavy chain (NHGMGGINSGAGSYTNYGAAVKGRATSVYGGSISLITDVIDT) and the light chain variable region (SG—SYSN--YYGENNNRPGGYDDSTDSGI) with the IgY framework region DNA fragment sequence, and add EcoRI restriction site sequence and protection base at the front end of the DNA fragment respectively, and add BamHI restriction site sequence and protection base at the end; then perform total gene synthesis.

[0171] Step 2), loading the DNA fragment into the eukaryotic expression vector PTT5: The synthesized DNA fragment is digested with EcoRI and BamHI and then inserted between EcoRI / BamHI of the eukaryotic expression vector PTT5 to obtain a recombinant vector containing the recombinant chicken anti-human TK1 (5 # ) full-length IgY monoclonal antibody.

[0172] Example 3

[0173] This example provides a method for preparing cells of a recombinant vector containing a chicken-derived anti-human TK1 recombinant full-length monoclonal IgY antibody gene, which includes the following steps:

[0174] Step 1), adjust the cell density: Count the 293T suspension cells, centrifuge to discard the supernatant, and adjust the cell density to 4.0×10 6 cells / mL, and culture at 37°C with 5% CO2 by volume for 4 h.

[0175] Step 2), transfection: Add DNA (5 # )(μg): PEI (1 mg / mL) in a ratio of 1:4 to the transfection medium, vortex and mix well, and incubate at room temperature for 15 min; add the mixture dropwise to the cell suspension to be transfected, and gently shake while adding.

[0176] Step 3), cell culture: The transfected cells are cultured at 37°C with 5% CO2 by volume for 120 h, and then the supernatant is collected for purification.

[0177] Example 4

[0178] This embodiment provides a preparation method of a detection kit containing a recombinant full-length monoclonal IgY antibody against human TK1 derived from chicken:

[0179] The preparation methods of the calibration product, quality control product, blocking agent, magnetic particle reagent coupled with the first antibody, biotin-labeled second antibody, streptavidin-labeled alkaline phosphatase, luminescent substrate, anti-reagent, diluent and washing solution in the kit are as follows:

[0180] 1. Magnetic particle reagent coupled with the first antibody

[0181] 1) Put the fully mixed toluenesulfonyl magnetic particle concentrate into a reaction bottle. After all the magnetic particles are adsorbed by the magnetic field, aspirate the supernatant. Add 10 times the volume of magnetic particle activation buffer to the reaction bottle, shake and wash for 10 min, then place the reaction bottle in the magnetic field for 15 min, and aspirate the supernatant; repeat the washing of the magnetic particles 2 times; finally, dilute the magnetic particle solution to 1 - 30 mg / ml, mix well and set aside for use;

[0182] 2) For the coupling reaction, add the first antibody (recombinant chicken anti-human TK1 full-length IgY monoclonal antibody) to the toluenesulfonyl magnetic particle solution prepared in step 1) according to the mass ratio of toluenesulfonyl magnetic particle solution: first antibody of 100:1, add 1 / 10 of the total volume of magnetic particle catalytic buffer, and keep mixing at 37 °C for 18 h;

[0183] 3) Add 1 / 20 of the total volume of the solution of 10% BSA to the magnetic particle solution prepared in step 2), and keep mixing at 37 °C for 6 h;

[0184] 4) Place the reaction bottle in the magnetic field for 15 min. After the toluenesulfonyl magnetic particles are adsorbed to the magnetic field, wash 3 times with the magnetic particle washing solution, and then make up the volume to 1 - 20 mg / ml and store at 4 °C to obtain the magnetic particle-coupled first antibody.

[0185] Among them, the preparation method of the magnetic particle activation buffer is to dissolve 1 - 10 g of boric acid in 900 ml of deionized water, adjust the pH to 8 - 10 with NaOH, make up the volume to 1 L and filter with a 0.45 μm filter membrane;

[0186] The preparation method of the magnetic particle catalytic buffer is: dissolve 30 - 300 g of ammonium sulfate in 1 L of magnetic particle activation buffer, and filter with a 0.45 μm filter membrane after complete dissolution;

[0187] The magnetic particle washing solution is a TBST buffer with a pH of 7.4.

[0188] 2. Biotin-labeled second antibody

[0189] Prepare a 0.1 - 20 mg secondary antibody solution from the recombinant chicken anti - human TK1 full - length IgY monoclonal antibody using PBS. Prepare a 5 - 50 mg solution of biotin using DMSO. Add the biotin solution to the antibody solution and mix well. Incubate on ice for 2 h or react at room temperature for 30 min to obtain a biotin - labeled secondary antibody solution.

[0190] 3. Luminescent substrate

[0191] Alkaline phosphatase luminescent substrate No. 1, product number: APSUB - 1 (Beijing Aivide Biotechnology Co., Ltd.).

[0192] 4. Blocking agent

[0193] The non - fat milk powder is provided by Thermo Fisher Scientific (China) Co., Ltd.

[0194] 5. Diluent

[0195] Dissolve 0.1 g - 10 g of the blocking agent in 1 L of Tris buffer, add 0.1 - 5 mL of preservative, and filter through a 0.22 μm filter membrane after complete dissolution to prepare it.

[0196] 6. Biotinylated antibody working solution

[0197] Dilute the biotinylated anti - thymidine kinase 1 - IgY polyclonal antibody to a final concentration of 0.05 - 15 μg / mL.

[0198] 7. Wash solution

[0199] pH 7.4 TBST buffer (30 - fold concentrated solution)

[0200] 8. Streptavidin - labeled alkaline phosphatase

[0201] The streptavidin - labeled alkaline phosphatase is purchased from Invitrogen streptavidin - labeled alkaline phosphatase and is prepared by diluting it 50,000 - fold with the diluent.

[0202] Example 5

[0203] This example provides a preparation method of a drug containing a chicken - derived anti - human TK1 recombinant full - length monoclonal IgY antibody:

[0204] 1. Construction of the recombinant chicken anti - human TK1 full - length IgY gene double - expression cassette expression vector: Using 5 #The amino acid sequences of the heavy and light chains of the antibody were optimized into genes synthesized with CHO codons. HindⅢ and XmaⅠ restriction enzyme sites were designed at both ends of the heavy chain, and BsiWⅠ and EcoRⅠ restriction enzyme sites were designed at both ends of the light chain. The heavy chain was inserted into the pEE 6.4 vector with the designed restriction enzyme sites, and the light chain was inserted into the pEE 12.4 vector with the designed restriction enzyme sites. The two expression vectors were digested with NotⅠ and SalⅠ, and the heavy chain ORF in pEE 6.4 was recovered and ligated and inserted into pEE 12.4, and then linearized with PVUⅠ.

[0205] 2. Transfection and pressure application of CHO cells: The CHO cells were revived and the cell state was adjusted to the best. The linearized expression vector was transfected into the cells by electroporation using the GenePulserXcell™ system, and the medium MSX was supplemented the next day for three weeks of pressure application. After most of the cells died, new clone clusters grew. Thirty high-expressing cell wells were selected and transferred to a 24-well plate, and then transferred to a 6-well plate. The cells were cryopreserved after the cell density reached 80%.

[0206] 3. Screening of monoclonal cell lines: Applying pressure in small gradients multiple times is beneficial for obtaining high-expressing cell lines, but the expression of the cell lines is unstable. After removing the screening pressure, the expression level often decreases, preparing for the next step of screening by the limiting dilution method. The pressure application concentration can be flexibly changed according to the actual culture situation. It is tentatively set to apply pressure three rounds, which can be adjusted according to the actual situation.

[0207] Screening by the limiting dilution method: Exploration of cell seeding density: Starting from a density of 1000 cells / well, serial dilution was performed. Ten plates were seeded at each density and cultured at 37℃ with 5% CO2 by volume, and observed. The supernatant of the 96-well plate was taken to detect ELISA, and the clone numbers with high titers were selected and cultured in a 12-well plate. The supernatant of the 12-well plate was taken to detect ELISA, and the clone numbers with high titers were selected and cultured in a 6-well plate. The supernatant of the 6-well plate was taken to detect ELISA, and the clone numbers with high titers were selected and cultured in a shake flask. The cell state was comprehensively evaluated by microscopic observation, cell density, and viability. When the viability was greater than 95%, 10 - 20 vials of the cell line were cryopreserved; 1.5 - 2.0 mL of the supernatant was taken to detect ELISA and HPLC to evaluate antibody expression, and HPLC was used to evaluate the final antibody yield (standard ≥5 g / L).

[0208] 4. Observation of stability:

[0209] At each culture passage when the cell viability dropped below 50%, 1.5 - 2.0 mL of the supernatant was taken to detect ELISA and HPLC to evaluate antibody expression, and HPLC was used to evaluate the final antibody yield. It was required that the final yield decreased by less than 10%. If the requirements were met, a cell bank was established.

[0210] 5. Fermentation and purification of the stable cell line.

[0211] Select cell lines with a yield of more than 5 g / L for fermentation production. Purify and remove impurities from the cell supernatant to ensure that the product meets the antibody drug standards.

[0212] This application uses an improved phage display method and the C-terminal 195-225 polypeptide of human TK1 to develop a chicken anti-human TK1 full-length-IgY monoclonal antibody. Using the improved phage display method, the positive clone rate is as high as 98%. A super-large library of 3.4×10 9 pfu / ml was rapidly constructed within 3 weeks, which can completely prevent environmental phage contamination. Therefore, we successfully obtained single-chain antibodies that meet the requirements of the inventors and obtained the sequences of the variable regions of the heavy and light chains of the antibody. After analyzing the obtained antibody fragments, the full-length IgY antibody was constructed, and then the antibody was expressed through a eukaryotic expression system and purified. The high specificity and high sensitivity of the antibody were verified by detection methods such as ELISA titer, immunoblotting, and immunohistochemistry.

[0213] More importantly, serological identification pointed out that using the two serum detection kits assembled by us for serum detection, the detection results showed that the correlation coefficient of the two methods was as high as r = 0.857. In the future, the fully automated chemiluminescence analyzer will replace the past semi-automatic enhanced chemiluminescence immunoassay system, enabling the new generation of TK1-IgY recombinant antibody kit to run on the fully automated instrument, which can meet the detection needs of large-scale early tumor risk screening. At present, the TK1-IgY-rmAb has been successfully constructed using phage display library technology and run on the new fully automated instrument, which has not been reported yet and is the first in the world.

[0214] To confirm that the chicken-derived anti-human TK1 recombinant full-length monoclonal IgY antibody (hTK1-IgY-rmAb) can replace the chicken anti-human TK1 natural full-length IgY polyclonal antibody (hTK1-IgY-pAb), the inventors used hTK1-IgY-rmAb and hTK1-IgY-pAb to detect the concentration of STK1p in 95 voluntary blood donors (self-reported no tumors or infectious diseases) by enhanced chemiluminescence dot blot (ECL-dot blot). As Figure 5 shown, the coincidence rate of the Pearson test was r = 0.988, confirming that it is reliable to replace hTK1-IgY-pAb with hTK1-IgY-rmAb.

[0215] The collection and use of serum and tissue specimens in this application were carried out in accordance with the Declaration of Helsinki in 1964 and the tripartite guidelines of the International Conference on Harmonization of Clinical Practice. This study was approved by the Changzhou Cancer Hospital, with the ethics number 2020-SY-032. All participants provided informed consent before entering the study.

[0216] The above-described embodiments are only preferred embodiments of the present invention, which are used to explain the present invention rather than to limit the scope of the present invention. The invention name of the present invention has been described through specific embodiments. Those skilled in the art can appropriately change links such as raw materials and conditions by referring to the content of the present invention to achieve corresponding other purposes, and their related changes do not depart from the content of the present invention. All similar substitutions and modifications are obvious to those skilled in the art and are regarded as being included within the scope of the present invention.

Claims

1. A single-chain antibody of chicken-derived anti-human TK1 recombinant IgY, characterized in that: The single-chain antibody is composed of a heavy chain, a linker peptide and a light chain. The variable region of the single-chain antibody includes HCDR1-3 of the heavy chain and LCDR1-3 of the light chain. The amino acid sequence of HCDR-1 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR-2 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR-3 is shown in SEQ ID NO: 8, the amino acid sequence of LCDR-1 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR-2 is shown in SEQ ID NO: 19, and the amino acid sequence of LCDR-3 is shown in SEQ ID NO:

23.

2. A chicken-derived anti-human TK1 recombinant full-length monoclonal IgY antibody, characterized in that: The amino acid sequence of the chicken-derived recombinant full-length monoclonal IgY antibody against human TK1 is SEQ ID NO:

28.

3. A recombinant vector, characterized in that: The recombinant vector contains a nucleic acid encoding the amino acid sequence recited in claim 1 or 2, and the recombinant vector is constructed using the PTT5 plasmid.

4. A cell containing the recombinant vector, characterized in that: The cell contains the recombinant vector recited in claim 3, and a stable cell line is constructed using Chinese hamster ovary (CHO) cells.

5. A test card, test strip or test kit, characterized in that: The test card, test strip or test kit contains the antibody recited in any one of claims 1 or 2.

6. A drug, characterized in that: Contains the antibody recited in any one of claims 1 or 2.

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