Application of antibody fragment-enzyme conjugate in high-throughput sequencing

Abstract

The invention discloses an application of an antibody fragment-enzyme conjugate with small molecular weight in high-throughput sequencing. The invention provides a high-throughput sequencing method, which is characterized in that specific recognition is carried out on labeled dNTP by using an antibody fragment without being limited by steric hindrance and complexity of a fragment three-dimensional structure, so that the sequencing quality is improved; runon / Lag and the base mismatch rate caused by a complex three-dimensional structure in the sequencing process can be effectively reduced. The molecular weight of protease used in the sequencing process is effectively reduced, so that the reduction of the sequencing quality caused by steric hindrance of protein in long-read-long sequencing can be reduced, the sequencing quality is effectively ensured, and the accuracy of sequencing data is improved.

Description

Technical Field

[0001] The invention belongs to the field of high-throughput sequencing, relates to the application of an antibody fragment-enzyme conjugate in high-throughput sequencing, and specifically relates to the application of an antibody fragment-enzyme conjugate with a small molecular weight in high-throughput sequencing. Background Art

[0002] Sequencing by synthesis (SBS) technology is a new generation sequencing technology widely used worldwide. It usually uses four different fluorescent labeled bases (dNTPs) as substrates and single-stranded DNA of the sample to be tested as a template to simulate the DNA replication process. When each dNTP hybridizes to the sample to be tested, it will present different fluorescent signals, and the base sequence in the sample can be identified based on the different fluorescent signals; after a round of sequencing is completed, the fluorescent dye will be cut and removed by the cutting reagent to start the next cycle. This sequencing has the characteristics of high throughput, high efficiency, and low cost. However, the dNTP labeled with fluorescent dyes may have residual residues in the elution step during the sequencing process, and these residues may affect the flexibility of the newly synthesized chain and the binding efficiency of the polymerase to the continuous extension of the chain and the fidelity of the polymerase, and may also partially quench the fluorescent signal in the next cycle, thereby affecting the quality of sequencing. Therefore, the fluorescent dye can be modified to the base antibody that can specifically recognize the base, so that the high-natural dNTP can be effectively used for polymerization reaction, and the sensitivity of the sequencing reaction can be improved. However, the antibodies commonly used have a relatively large molecular weight. As sequencing proceeds, it becomes increasingly difficult for the huge antibodies to shuttle and specifically recognize and bind to the fragment to be tested, resulting in a decrease in sequencing quality.

[0003] At present, the equipment that uses fluorescence signals for sequencing usually needs to be equipped with different laser excitations and filters of different wavelengths for signal collection, which makes the optical system of the equipment bulky and requires a matching condensation constant temperature system. The laser and detector will continue to age and lose value as the use time increases, resulting in an irreversible decline in sequencing quality. Therefore, a new signal generation method has gradually emerged, that is, using chemiluminescent signals instead of traditional fluorescent signals, which can output light signals under specific conditions and distinguish different bases. This method does not require a specially prepared laser light source for excitation, avoids the use of a large optical system, and breaks through traditional sequencer equipment in terms of size and convenience. At the same time, this method also has the characteristics of simple signal recognition, rapid biochemical reactions, and high sequencing throughput, and is an important sequencing method. The chemiluminescence methods currently used are mostly enzymatic chemiluminescence, and a large number of luciferases and luciferin substrates will be used in the sequencing system; for high-throughput long-read sequencing, there are extremely high requirements for the stability of enzymes and substrates; therefore, using traditional chemiluminescence methods, different combinations of bases, antibodies, etc. can be labeled, and the base sequence can be judged by base signal intensity and base luminescence dynamics (response time, luminescence lifetime, half-life) during the sequencing process. This combination can reduce the mutual interference between the two detections during the sequencing process, thereby reducing the base misreading caused by signal confusion and improving the accuracy of base recognition. Bioluminescence detection technology based on luciferases such as firefly luciferase, bacterial luciferase, sea renilla luciferase, Gaussia luciferase (Gaussia luciferase, GLuc), Nanoluc luciferase (Nluc), and shrimp luciferase has become a research hotspot in this field.

[0004] Currently, the signal factor used in the platform based on enzymatic chemiluminescence sequencing is a chemical conjugate of antibody and luciferase. The specific recognition effect of the antibody is used to distinguish the bases, and the catalytic effect of luciferase is used to generate light signals at the positions of the bases, thereby distinguishing the signal output of the bases. Summary of the invention

[0005] As mentioned above, due to the technical problems in the prior art such as "due to the large molecular weight of the signal factor used in sequencing (>180kDa), as the sequencing time / length increases, the difficulty of the signal factor shuttling through the specific binding DNB increases continuously, accompanied by an increase in nonspecific adsorption during the sequencing process and an increase in the difficulty of elution; therefore, the sequencing index decreases significantly as the sequencing is prolonged, and the sequencing time required for a single cycle is also prolonged", the purpose of the present invention is to develop a small protein based on a fragment antibody-luciferase conjugate, which can not only specifically identify bases during the sequencing process, but also utilize the small size advantage of the protein molecule itself to efficiently shuttle during the sequencing process, effectively reducing the Lag / Runon and base mismatch rate caused by steric hindrance during the sequencing process.

[0006] In a first aspect, the present invention claims a method for high-throughput sequencing, which can improve sequencing quality.

[0007] The high-throughput sequencing method claimed in the present invention comprises the following steps: in a DNA amplification reaction of high-throughput sequencing, the first of the four dNTPs is labeled with substance 1, the second is labeled with substance 2, the third is labeled with the substance 1 and the substance 2, and the fourth is not labeled; then, the four dNTPs are distinguished by using a substance 1'-luciferase 1 conjugate and a substance 2'-luciferase 2 conjugate;

[0008] The substance 1' can specifically bind to the substance 1; the substance 2' can specifically bind to the substance 2; at least one of the substance 1 and the substance 2 is an antigen or a hapten molecule, and correspondingly at least one of the substance 1' and the substance 2' is an antibody fragment that can specifically recognize the antigen or hapten molecule.

[0009] In some embodiments, a portion of the third of the four dNTPs is labeled with the substance 1, and the other portion is labeled with the substance 2. In some embodiments, half of the third of the four dNTPs is labeled with the substance 1, and the other half is labeled with the substance 2.

[0010] In some embodiments, any one of the substance 1 and the substance 2 is an antigen or hapten molecule, and the other is a non-antigen or hapten molecule. In a further embodiment, any one of the substance 1 and the substance 2 is biotin (corresponding to a non-antigen or hapten molecule), and accordingly any one of the substance 1' and the substance 2' is streptavidin; the other of the substance 1 and the substance 2 is digoxin (corresponding to an antigen or hapten molecule), and accordingly the other of the substance 1' and the substance 2' is an anti-digoxin antibody fragment. In some embodiments, the substance 1 and the substance 2 are different antigens or hapten molecules, and accordingly, the substance 1' is an antibody fragment that specifically recognizes the substance 1, and the substance 2' is an antibody fragment that specifically recognizes the substance 2.

[0011] In the above method, luciferase 1 corresponds to substrate 1, and luciferase 2 corresponds to substrate 2. By sequentially introducing substrates of different luciferases for luminescence detection, taking pictures in sequence can achieve the distinction, differentiation, recognition or identification of 4 different bases, thereby achieving the purpose of sequencing. In some embodiments, in the luminescence detection process of high-throughput sequencing, it is assumed that the first picture is taken after the first introduction of substrate 1, and the second picture is taken after the second passage of substrate 2. Combining the results of the two pictures, only the first picture detects the corresponding first dNTP of the luminescence signal in the previous text; only the second picture detects the corresponding second dNTP of the luminescence signal in the previous text; the first picture and the second picture both detect the corresponding third dNTP of the luminescence signal in the previous text; the first picture and the second picture do not detect the corresponding fourth dNTP of the luminescence signal in the previous text.

[0012] In some embodiments, the luciferase 1 and the luciferase 2 are selected from firefly luciferase, bacterial luciferase, Renilla luciferase, Gaussia luciferase, Nanoluc luciferase and spiny shrimp luciferase, respectively. In some embodiments, the luciferase 1 is Gaussia luciferase, and the luciferase 2 is Nanoluc luciferase.

[0013] In the above method, the conjugate formed by the antibody fragment and luciferase is obtained by any of the following methods:

[0014] (A1) cutting the full-length antibody that specifically recognizes the antigen or hapten molecule into the antibody fragment, and then connecting the antibody fragment to luciferase by chemical coupling;

[0015] (A2) directly constructing a fusion plasmid for expressing the antibody fragment and luciferase conjugate by biosynthesis, and then introducing the fusion plasmid into a recipient cell or recipient bacteria for expression;

[0016] (A3) The antibody fragment is biosynthesized, and then the antibody fragment is linked to luciferase by chemical coupling.

[0017] In some embodiments, in (A1) and (A3), when the chemical coupling is performed, the antibody fragment exposes the sulfhydryl group by the action of a reducing agent or a non-reducing agent, the luciferase is activated using a coupling agent, and the treated antibody fragment and the activated luciferase are linked with or without the addition of a linker.

[0018] In some embodiments, the antibody fragment exposes the sulfhydryl group by the action of a reducing agent, wherein the reducing agent is selected from dithiothreitol, tris(2-carbonylethyl)phosphine hydrochloride and mercaptoethylamine; preferably, the reducing agent is tris(2-carbonylethyl)phosphine hydrochloride (TECP).

[0019] In some embodiments, the coupling agent is selected from 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt, and 2-iminothiolane hydrochloride; preferably, the coupling agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt (Sulfo-SMCC).

[0020] In some embodiments, the treated antibody fragment and the activated luciferase are linked under the condition of adding a linker, and the linker is bismaleimide or maleimide-polyethylene glycol-maleimide.

[0021] In some embodiments, the treated antibody fragment and the activated luciferase are linked without the addition of a linking agent.

[0022] In the above method, the antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, and a F(ab')2 fragment. In some embodiments, the antibody fragment is a Fab fragment or a F(ab')2 fragment.

[0023] In some embodiments, the substance 1 is biotin, the substance 1' is streptavidin; the substance 2 is digoxin, and the substance 2' is a Fab fragment or F(ab')2 fragment that specifically recognizes digoxin. That is, the substance 1'-luciferase 1 conjugate is a conjugate formed by streptavidin and Gaussia luciferase (GLuc luciferase); the substance 2'-luciferase 2 conjugate is a conjugate formed by a Fab fragment or F(ab')2 fragment that specifically recognizes digoxin and Nanoluc luciferase.

[0024] In a second aspect, the present invention claims the application of the method described in the first aspect above in any of the following:

[0025] (B1) Improving the accuracy of sequencing data from high-throughput sequencing;

[0026] (B2) Reducing Lag and / or Runon during high-throughput sequencing;

[0027] (B3) Reduce the base mismatch rate during high-throughput sequencing.

[0028] In a third aspect, the present invention claims protection for the use of the method described in the first aspect above in the preparation of products of high-throughput sequencing technology.

[0029] The product may be a reagent, a test kit or a complete set of products.

[0030] In a fourth aspect, the present invention claims protection for a set of products for high-throughput sequencing.

[0031] The complete set of products for high-throughput sequencing claimed in the present invention comprises the following (C1)-(C3):

[0032] (C1) the four dNTPs described in the first aspect above;

[0033] (C2) the substance 1'-luciferase 1 conjugate described in the first aspect above;

[0034] (C3) The substance 2'-luciferase 2 conjugate described in the first aspect above.

[0035] In some embodiments, the kit further comprises (C4) substrate 1 corresponding to the luciferase 1, and (C5) substrate 2 corresponding to the luciferase 2.

[0036] In a fifth aspect, the present invention claims protection for the use of the set of products described in the fourth aspect above in high-throughput sequencing.

[0037] In the above aspects, the high-throughput sequencing is second-generation sequencing, especially long-read sequencing.

[0038] In a sixth aspect, the present invention claims a method for preparing an antibody fragment-luciferase conjugate.

[0039] The method for preparing the antibody fragment-luciferase conjugate claimed in the present invention may include any of the following steps:

[0040] (A1) cutting the full-length antibody that specifically recognizes the antigen or hapten molecule into the antibody fragment, and then connecting the antibody fragment to luciferase by chemical coupling;

[0041] (A2) directly constructing a fusion plasmid for expressing the antibody fragment and luciferase conjugate by biosynthesis, and then introducing the fusion plasmid into a recipient cell or recipient bacteria for expression;

[0042] (A3) The antibody fragment is biosynthesized, and then the antibody fragment is linked to luciferase by chemical coupling.

[0043] In some embodiments, in (A1) and (A3), when the chemical coupling is performed, the antibody fragment exposes the sulfhydryl group by the action of a reducing agent or a non-reducing agent, the luciferase is activated using a coupling agent, and the treated antibody fragment and the activated luciferase are linked with or without the addition of a linker.

[0044] In some embodiments, the antibody fragment exposes the sulfhydryl group by the action of a reducing agent, wherein the reducing agent is selected from dithiothreitol, tris(2-carbonylethyl)phosphine hydrochloride and mercaptoethylamine; preferably, the reducing agent is TECP.

[0045] In some embodiments, the coupling agent is selected from 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt, 2-iminothiolane hydrochloride; preferably, the coupling agent is Sulfo-SMCC.

[0046] In some embodiments, the treated antibody fragment and the activated luciferase are linked under the condition of adding a linker, and the linker is bismaleimide or maleimide-polyethylene glycol-maleimide.

[0047] In some embodiments, the treated antibody fragment and the activated luciferase are linked without the addition of a linking agent.

[0048] Further, the antibody fragment is selected from Fab fragment, Fab' fragment, F(ab')2 fragment. In some embodiments, the antibody fragment is a Fab fragment or a F(ab')2 fragment, preferably, the antibody fragment is a Fab fragment or a F(ab')2 fragment that specifically recognizes digoxin.

[0049] In some embodiments, the luciferase is selected from the group consisting of firefly luciferase, bacterial luciferase, Renilla luciferase, Gaussia luciferase, Nanoluc luciferase and spiny shrimp luciferase, preferably, the luciferase is Nanoluc luciferase.

[0050] In a seventh aspect, the present invention claims protection for the antibody fragment-luciferase conjugate prepared by the method described in the sixth aspect above.

[0051] The key technology of the present invention is to use antibody fragments to specifically recognize labeled dNTPs without being limited by their steric hindrance and the complexity of the three-dimensional structure of the fragments; it can effectively reduce the Runon / Lag and base mismatch rates caused by the complex three-dimensional structure during sequencing and improve the sequencing quality.

[0052] The technology of the present invention can reduce the degradation of sequencing quality caused by the steric hindrance of proteins in long-read sequencing by effectively reducing the molecular weight of the protease used in the sequencing process. It can effectively improve the sequencing quality, effectively ensure the sequencing quality in long-read sequencing, and thus improve the accuracy of sequencing data. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of the preparation of whole antibody, Fab and F(ab')2 chemically coupled to luciferase.

[0054] Figure 2 This is a gel image of Fab / F(ab')2 cut by the kit. The sample order is: 1# is the crude product of F(ab')2 cut; 2# is the crude product of Fab cut; 3# is the purified F(ab')2; 4# is the purified Fab.

[0055] Figure 3 The gel images of Fab fragments treated with different reducing agents. The sample order is: 1# is Fab treated with Traut's reagent; 2# is Fab reduced with DTT; 3# is Fab reduced with TECP; 4# is Fab reduced with MEA.

[0056] Figure 4 The gel images of luciferase treated with different reagents. The order of samples is: 1# is luciferase treated with Traut's reagent; 2# is luciferase; 3# is luciferase treated with Sulfo-SMCC.

[0057] Figure 5 The gel images of the conjugates under different conditions. The sample order is: 1# is F(ab')2-luciferase; 2# is Fab-luciferase; 3# is F(ab')2-Mal-PEG-Mal-luciferase; 4# is Fab-Mal-PEG-Mal-luciferase; 5# is F(ab')2-Mal-C12-Mal-luciferase; 6# is Fab-Mal-C12-Mal-luciferase.

[0058] Figure 6 The enzymatic chemiluminescence intensity of the conjugates obtained under different conditions is detected. Figure 5 The naming in the table corresponds to that in the table, that is, 1 corresponds to 1#, 2 corresponds to 2#, and so on.

[0059] Figure 7 The protein band diagram of each component in the protein preparation process. The sample order is: 1# is the full-length antibody of digoxin; 2# is the crude product after cleavage; 3# is the purified Fab; 4# is the Fab reduced by reducing agent; 5# is the Nanoluc luciferase after activation treatment; 6# is the coupling product.

[0060] Figure 8 The Q30 of the experimental group (Fab fragment) and the control group in PE150 is compared. The concentrations of the conjugates added to the experimental group 1 and the experimental group 2 are different. The working concentration of the experimental group 1 is 6 μg / mL; the working concentration of the experimental group 2 is 9 μg / mL.

[0061] Fig. 9 Comparison of Runon / Lag between the experimental group (Fab fragment) and the control group in PE150. The concentrations of the conjugates added to experimental group 1 and experimental group 2 were different, with the working concentration of experimental group 1 being 6 μg / mL and the working concentration of experimental group 2 being 9 μg / mL.

[0062] Fig.10 The protein band diagram of each component in the protein preparation process. The sample order is: 1# is F(ab')2; 2# is the reduced F(ab')2 fragment; 3# is the purified reduced F(ab')2 fragment; 4# is the activated Nanoluc luciferase; 5# is the purified activated Nanoluc luciferase; 6# is the coupling product; 7# is the pure luciferase; 8# is the digoxin antibody.

[0063] Fig.11 Comparison of Q30 between the control group and the experimental group (F(ab')2 fragment) in PE150.

[0064] Fig.12 Comparison of Runon / Lag between the experimental group (F(ab')2 fragment) and the control group in PE150.

[0065] Fig.13 The results are for the base mismatch rate determination of the control group and the experimental group (F(ab')2 fragment) in PE150.

[0066] Fig.14 Schematic diagram of the preparation of small signaling factors by chemical coupling of cell-expressed antibody fragments to luciferase.

[0067] Fig.15 Exemplary structures of different antibody fragments expressed by fusion with luciferase. DETAILED DESCRIPTION

[0068] The present invention relates to the use of antibody fragments to specifically recognize labeled dNTPs. After the fragments are chemically coupled with luciferase, a small molecular weight antibody fragment-enzyme conjugate can be obtained. When dNTPs are specifically recognized (antigens are modified on dNTPs, luciferase is modified on antibodies, and the specific binding of antibody fragments to antigens allows luciferase to indirectly label dNTPs), an output light signal is generated and detected, thereby completing gene sequencing on a luminescent platform.

[0069] The antibody fragments used in the present invention are Fab and F(ab')2 with specific recognition sites. The fragments are prepared by enzymatic digestion with specific enzymes or by biological expression; then the antibody fragment-luciferase conjugate is obtained by chemical coupling or biological expression.

[0070] In the chemical coupling scheme, the prepared antibody fragment will be exposed to stable sulfhydryl groups through the action of reducing agents (such as dithiothreitol (DTT), tris (2-carbonylethyl) phosphine hydrochloride (TECP), mercaptoethylamine (MEA)) or non-reducing agents (such as 2-iminothiolane hydrochloride Traut's reagent, etc.); luciferase will be activated by coupling agents (4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (sulfo-SMCC), 2-iminothiolane hydrochloride (Traut's reagent)) and the like; the two components after treatment are fully reacted without adding a linker or with adding a linker (such as bismaleimide, maleimide-polyethylene glycol-maleimide); and the reaction product is quality inspected, and after the detection, the antibody fragment-luciferase conjugate is sequenced and tested. The exemplary technical solutions are as follows Figure 1 shown.

[0071] In the biological fusion expression scheme, new plasmids with different site modifications, different linker fragments, and different modification ratios can be constructed based on the known Fab / F(ab')2 fragments and the plasmid information of the target luciferase, and then the plasmids are transfected and expressed to obtain the target signal factor (i.e., antibody fragment-luciferase conjugate).

[0072] Specifically, antibody fragment-luciferase conjugates can be prepared by the following three methods: (1) Under the premise of ensuring the specific recognition site of the antibody, the full-length antibody is cut into antibody fragments (such as Fab / F(ab')2 fragments), for example, by enzyme cutting, and then the antibody fragment is coupled with luciferase by chemical coupling to prepare the antibody fragment-luciferase conjugate; (2) by constructing a fusion plasmid of antibody fragment (such as Fab / F(ab')2 fragment) and luciferase, the antibody fragment-luciferase conjugate is directly expressed; (3) by biosynthesizing antibody fragments (such as Fab / F(ab')2 fragments) and then coupling with luciferase by chemical coupling to prepare the antibody fragment-luciferase conjugate. The antibody fragment-luciferase conjugate prepared by the above three methods can effectively reduce Lag / Runon, base error rate, etc. in the sequencing process as a signal factor; and can greatly reduce the nonspecific adsorption of the signal factor in the sequencing process, and can shorten the excision time in the sequencing process.

[0073] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are exemplary and are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0074] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0075] The sources of reagents and instruments in the following examples are shown in Table 1.

[0076] Table 1. Sources of reagents and instruments

[0077] Reagent / Instrument Name Manufacturer / Brand Antibody Company's own production <![CDATA[Fab / F(ab’)2 Preparation Kit]]> Thermo Fisher Sulfo-SMCC Thermo Fisher Traut's reagent Maclean TECP Shanghai Bioengineering DTT Shanghai Bioengineering <![CDATA[Mal-PEG 5000 -times]]> Xi'an Ruixi Mal-C12-Mal Maclean 20×PBS Shanghai Bioengineering 50mL ultrafiltration tube (10k) Millipore ELISA reader Perkin Elemer DTNB Shanghai Bioengineering

[0078] Example 1: Preparation of small molecular weight antibody fragment-enzyme conjugates by chemical coupling method and application of the prepared antibody fragment-enzyme conjugates in high-throughput sequencing

[0079] 1. Preparation of antibody fragment (Fab / F(ab')2) and enzyme conjugates by chemical coupling method

[0080] 1. Use the Fab / F(ab')2 fragment preparation kit purchased from Thermo Fisher Scientific to cut 0.5mL of digoxin full-length antibody (Qingdao BGI Intelligent Manufacturing Puhui Technology Co., Ltd., concentration is 4mg / mL) according to the instructions. The Fab cutting condition is 37℃4h; the F(ab')2 cutting condition is 37℃28h. The crude product is purified by Protein A column, and the product band diagram is as follows Figure 2 shown.

[0081] 2. Antibody fragment thiolation

[0082] (1) The antibody fragments were reduced using a reducing agent to expose the thiol groups. The reducing agents DTT, TECP, and MEA were used respectively; the reduction conditions were: DTT: 5 mM, RT, 30 min; TECP: 50 mM, RT, 30 min; MEA: 10 mM, 37°C, 90 min. The fragments obtained by reduction are as follows Figure 3 As shown in 2# to 4#.

[0083] (2) The antibody fragment was reacted with a non-reducing agent to expose the sulfhydryl group. The antibody fragment was treated with 10 mM Traut's reagent at room temperature for 30 min, and the resulting -SH was detected by DTNB. The sample gel was as follows: Figure 3 As shown in 1#.

[0084] Combining the reducibility, selectivity, repeatability, and convenience of post-processing of the above three reducing agents and non-reducing agents, we chose TECP as the reducing agent for subsequent experiments.

[0085] 3. Luciferase activation

[0086] (1) The amino group of Nanoluc luciferase (Qingdao HuaDa Intelligent Manufacturing Technology Co., Ltd., 2 mg / mL) was reacted with the coupling agent SMCC (2 equivalents) at room temperature for 30 min. The amino group and maleimide were connected to expose 1-2 succinimidyl esters of Nanoluc luciferase.

[0087] (2) Traut's reagent is used to chemically react the amino group of luciferase to expose multiple -SH groups.

[0088] The gel image after the reaction is as follows Figure 4 shown.

[0089] 4. Connect

[0090] For the samples in steps 2 and 3, after the treatment time, ultrafiltration was performed using a 10k ultrafiltration tube with 1× PBS for 10 washes. 4times. After washing, the reaction systems in steps 2 and 3 were fixed to 500 μL. For the reagent pair of TECP reduction-Sulfo-SMCC activation treatment, no additional linker is added. The samples of equal volume are directly mixed thoroughly and placed in a 4°C refrigerator overnight for reaction; for the reagent pair of Traut's reagent treatment-Traut's activation, the two samples are mixed in equal volumes, and then the linker (linker Mal-PEG5000-Mal, Mal-C12-Mal; the working concentration of each is 10 μg / mL) is added and mixed thoroughly, and the reaction is placed in a 4°C refrigerator overnight.

[0091] The obtained conjugate gel is shown in Figure 5 As shown in the above figure, the bismaleimide containing C12 has a poor connection effect due to its solubility; when using bis-Mal reagents for connection, the molecular weight of the coupling product is still relatively large (>140kDa) due to its lack of selectivity.

[0092] 5. Property screening

[0093] The properties of the coupled fragments obtained above were screened using the chemiluminescence detection module of the microplate reader (the sample numbering order was the same as in step 4); 100 μL of luciferase substrate (fluorocoelenterazine, 50 μM) was used as the substrate, 10 μg of the coupled fragments was used as the catalytic reagent, and the chemiluminescence intensity was recorded. The luminescence intensity statistical graph obtained is as follows Figure 6 shown.

[0094] Combined with the above results, we found that the samples treated with TECP / sulfo-SMCC maintained better luciferase activity and were more convenient to operate, so we will continue to use this system in the following experiments.

[0095] 2. Application of antibody fragment (Fab / F(ab')2) and enzyme conjugates in high-throughput sequencing

[0096] 1. Preparation of conjugates of Fab fragments and enzymes

[0097] (1) Use the Pierce Fab fragment preparation kit purchased from Thermo Fisher Scientific, take 2 mg of digoxin full-length antibody (Qingdao HuaDa Intelligent Manufacturing Puhui Technology Co., Ltd.) for cutting (see the instructions for specific operations); after the cutting is completed, use Protein A protein column to separate and purify to obtain the target fragment; the obtained target fragment is then treated with the reducing agent TECP to expose the sulfhydryl group; at the same time, the Nanoluc luciferase is activated by sulfo-SMCC; after the reaction of the above two systems is completed, use a desalting column and ultrafiltration tube for purification, and finally combine the tubes for connection reaction (see Example 1 for specific experimental steps). The detection diagram of the intermediates and target products during the preparation process is as follows Figure 7The molecular weight of the obtained conjugate is mainly concentrated in the 80-110kDa fragment area.

[0098] (2) Perform sequencing test on the conjugate using E5-PE150

[0099] The on-machine test cases of the present invention are mainly carried out using the BGI E-series sequencing platform, and the main technologies used are the DNA Nanoball (DNB) technology and the combined probe anchor polymerization technology (Combinatorial Probe-Anchor Synthesis, hereinafter referred to as cPAS) independently developed by BGI. The entire sequencing process is mainly divided into three parts: preparation of DNB; loading of DNB; sequencing. In simple terms, we will use the DNB kit to circularize the DNA library (Ecoli BGI standard library), and then use the obtained single-stranded circularized DNA as a template to perform rolling circle amplification under the action of DNA polymerase. 100-200 copies of the single-stranded circular DNA will form our target DNB. The specific process is as follows:

[0100] (a) Reagent preparation during sequencing

[0101] The one-step DNB preparation kit is produced by the company.

[0102] dNTP: produced by the company, dATP is labeled with biotin; dTTP is labeled with digoxigenin; dCTP is half labeled with biotin and half with digoxigenin; dGTP is not labeled except for the blocking group (azide).

[0103] Polymerase reaction solution: The system contains 20 μg / mL BG9 polymerase and 1 μM of each of the above dNTPs; the primer buffer is 50 mM Tri-HCl, 50 mM NaCl, 10 mM (NH4)2SO4, and 3 mM MgSO4.

[0104] Elution reaction solution: 5×SSC containing 0.05% (volume percentage) Tween 20.

[0105] Protein binding solution: TBST buffer, containing 1 μg / mL streptavidin (SA)-GLuc luciferase conjugate, 6 / 9 μg / mL antibody fragment conjugate (i.e., anti-digoxigenin antibody fragment Fab-Nanoluc luciferase conjugate prepared in the above steps).

[0106] Luciferase luminescent solution 1: 50mM fluoro-coelenterazine (Nanoluc luciferase corresponding substrate), 50mM Tri-HCl, 5mM NaCl.

[0107] Luciferase luminescent solution 2: 50mM coelenterazine (GLuc luciferase corresponding substrate), 50mM Tri-HCl, 5mMNaCl.

[0108] Note: Different enzymes have different specific recognition substrates. This substrate pairing can avoid cross-talk between them.

[0109] Excision reaction solution: 20 mM THPP, 0.5 M NaCl, 0.05% (volume percentage) Tween 20.

[0110] (b) Sequencing reaction

[0111] Polymerization: Pass 100 μL of polymerase reaction solution, adjust the polymerization temperature to 60°C, react for 3 minutes, allow the four dNTPs to polymerize and amplify onto DNBs, carefully remove the reaction solution, add 100 μL of elution reaction solution, wash away the solution phase of the reaction system in the previous step, and retain the dNTPs bound to the DNBs.

[0112] Binding: 100 μL of protein binding solution was introduced into the system and incubated at 60°C for 1 min. The small protein signal molecule (i.e., antibody fragment-luciferase conjugate) was fully bound to the DNB; the large protein (full-length antibody-luciferase conjugate) was used as a control experiment. That is, an experimental group and a control group were set up. Compared with the experimental group, the signal molecule used in the control group was a full-antibody-luciferin conjugate; after the same polymerization conditions were adopted, the full-length antibody-luciferase conjugate was added, and the same luminescent substrate and other conditions were used.

[0113] (c) Luminescence detection

[0114] 100 μL of luciferase luminescent solution 1 (40°C reaction) and luciferin luminescent solution 2 (25°C reaction) were passed in respectively, and the detection was performed while passing in. After luciferase luminescent solution 1 was passed in for 9 seconds, luciferin luminescent solution 2 was passed in for about 9 seconds, and 3 dark field photos and 9 luminescent photos of each were collected to record the luminescent signal. After the signal collection was completed, elution was performed with elution reaction solution.

[0115] Excision: Add 100 μL excision reaction solution, react at 60°C for 1 min, then remove the excision reaction solution; add 200 μL elution reaction solution for washing;

[0116] Repeat the above steps to proceed to the next cycle of sequencing.

[0117] (3) Results and analysis:

[0118] Base call software from BGI was used for base resolution, and the base sequence was compared with the genome sequence of E. coli. Figure 8The comparison of Q30 between the experimental group and the control group during the PE150 process was presented, and the results showed that the prepared small protein (i.e., antibody fragment-luciferase conjugate) ensured and improved the sequencing quality; Fig. 9 The Runon / Lag comparison during the entire sequencing process is shown. The small protein in the experimental group (i.e., antibody fragment-luciferase conjugate) can significantly reduce the Runon / Lag during the entire sequencing process. Low lag and low runon can make the sequencing read length longer and the sequencing quality higher.

[0119] 2. Preparation of conjugates of F(ab')2 fragments and enzymes

[0120] (1) Use the F(ab')2 fragment preparation kit purchased from Thermo Fisher Scientific, take 2.8 mg of digoxin full-length antibody (Qingdao HuaDa Intelligent Manufacturing Puhui Technology Co., Ltd.) for cutting (see the instructions for specific operations); after the cutting is completed, use Protein A protein column to separate and purify to obtain the target fragment; the obtained target fragment is then treated with the reducing agent TECP to expose the sulfhydryl group; at the same time, the Nanoluc luciferase is activated by sulfo-SMCC; after the reaction of the above two systems is completed, use a desalting column and ultrafiltration tube for purification, and finally combine the tubes for connection reaction. The detection diagram of the intermediates and target products during the preparation process is shown in the figure Fig.10 The molecular weight of the obtained conjugate is mainly in the range of 80-110 kDa.

[0121] (2) Conduct E25-PE150 test on the conjugate

[0122] The on-machine test cases of the present invention are mainly carried out using the BGI E-series sequencing platform, and the main technologies used are the DNA Nanoball (DNB) technology and the combined probe anchor polymerization technology (Combinatorial Probe-Anchor Synthesis, hereinafter referred to as cPAS) independently developed by BGI. The entire sequencing process is mainly divided into three parts: preparation of DNB; loading of DNB; sequencing. In simple terms, we will use the DNB kit to circularize the DNA library (Ecoli BGI standard library), and then use the obtained single-stranded circularized DNA as a template to perform rolling circle amplification under the action of DNA polymerase. 100-200 copies of the single-stranded circular DNA will form our target DNB. The specific sequencing steps are as follows:

[0123] (a) Reagent preparation during sequencing

[0124] The one-step DNB preparation kit is produced by the company.

[0125] dNTP: produced by the company, dATP is labeled with biotin; dTTP is labeled with digoxigenin; dCTP is half labeled with biotin and half with digoxigenin; dGTP is not labeled except for the blocking group (azide).

[0126] Polymerase reaction solution: The system contains 20 μg / mL BG9 polymerase and 1 μM of each of the above dNTPs; the primer buffer is 50 mM Tri-HCl, 50 mM NaCl, 10 mM (NH4)2SO4, 3 mM MgSO4.

[0127] Elution reaction solution: 5×SSC containing 0.05% (volume percentage) Tween 20.

[0128] Protein binding solution: TBST buffer, containing 1 μg / mL streptavidin (SA)-GLuc luciferase conjugate, 9 μg / mL antibody fragment conjugate (i.e., anti-digoxigenin antibody fragment F(ab')2-Nanoluc luciferase conjugate prepared in the above steps).

[0129] Luciferase luminescent solution 1: 50mM fluoro-coelenterazine (Nanoluc luciferase corresponding substrate), 50mM Tri-HCl, 5mM NaCl.

[0130] Luciferase luminescent solution 2: 50mM coelenterazine (GLuc luciferase corresponding substrate), 50mM Tri-HCl, 5mMNaCl.

[0131] Excision reaction solution: 20 mM THPP, 0.5 M NaCl, 0.05% (volume percentage) Tween 20.

[0132] (b) Sequencing reaction

[0133] Polymerization: Pass 100 μL of polymerase reaction solution, adjust the polymerization temperature to 60°C, react for 3 minutes, allow the four dNTPs to polymerize and amplify onto DNBs, carefully remove the reaction solution, add 100 μL of elution reaction solution, wash away the solution phase of the reaction system in the previous step, and retain the dNTPs bound to the DNBs.

[0134] Binding: 100 μL of protein binding solution was introduced into the system and incubated at 60°C for 1 min. The signal molecule containing the small protein (i.e., antibody fragment-luciferase conjugate) was fully bound to the DNB; the large protein (full-length antibody-luciferase conjugate) was used as a control experiment. That is, two parallel experiments were set up, one for the experimental group and the other for the control group. Compared with the experimental group, the signal molecule used in the control group was a full-antibody-luciferin conjugate; after the same polymerization conditions were adopted, the full-length antibody-luciferase conjugate was added, and the same luminescent substrate and other conditions were used.

[0135] (c) Luminescence detection

[0136] 100 μL of luciferase luminescent solution 1 (40°C reaction) and luciferin luminescent solution 2 (25°C reaction) were passed in respectively, and the detection was performed while passing in. After luciferase luminescent solution 1 was passed in for 9 seconds, luciferin luminescent solution 2 was passed in for about 9 seconds, and 3 dark field photos and 9 luminescent photos of each were collected to record the luminescent signal. After the signal collection was completed, elution was performed with elution reaction solution.

[0137] Excision: Add 100 μL excision reaction solution, react at 60°C for 1 min, then remove the excision reaction solution; add 200 μL elution reaction solution for washing;

[0138] Repeat the above steps to proceed to the next cycle of sequencing.

[0139] (3) Results and analysis:

[0140] Base call software from BGI was used for base resolution, and the base sequence was compared with the genome sequence of E. coli. Fig.11 The comparison of Q30 between the experimental group and the control group during the PE150 process was presented, and the results showed that the prepared small protein (i.e., antibody fragment-luciferase conjugate) ensured and improved the sequencing quality; Fig.12 The Runon / Lag comparison during the entire sequencing process is shown. The small protein in the experimental group (i.e., antibody fragment-luciferase conjugate) can significantly reduce the Runon / Lag during the entire sequencing process. Low lag and low runon can make the sequencing read length longer and the sequencing quality higher.

[0141] Fig.13 The mismatches of all bases in the entire sequencing process were compared, and it can be seen that the experimental group (9μg / mL) can significantly reduce the error rate of each type of base in the control group.

[0142] Example 2: Preparation of small molecular weight antibody fragment-enzyme conjugates by combining biosynthesis and chemical coupling methods and application of the prepared antibody fragment-enzyme conjugates in high-throughput sequencing

[0143] 1. Preparation of antibody fragment (Fab / F(ab')2) and enzyme conjugates by combining biosynthesis and chemical coupling

[0144] Fig.14 Schematic diagram of the preparation of small signaling factors by chemical coupling of cell-expressed antibody fragments to luciferase.

[0145] Fab and F(ab')2 antibody fragments (4 mg / mL, 1 mL) that specifically recognize digoxin were expressed by biosynthesis, and the Fab and F(ab')2 antibody fragments were reduced with 50 mM TECP solution for 30 min (see Example 1 for the specific reduction steps); Nanoluc luciferase was activated with sulfo-SMCC at room temperature for 30 min (see Example 1 for the specific activation steps); after the reaction was completed, the remaining small molecules in the reaction system were removed by ultrafiltration centrifugation with a 10Kda centrifuge tube, and the volumes of the two samples were finally concentrated to 1 mL. Then 1 mL of Fab / F(ab')2 was added to Nanoluc luciferase in 10 times, mixed thoroughly, and then placed in a 4°C refrigerator for overnight, and then the samples were characterized by SDS-PAGE.

[0146] The preparation of antibody fragments by biosynthesis effectively reduces the time cost of our product preparation and improves the sample yield.

[0147] 2. Application of antibody fragment (Fab / F(ab')2) and enzyme conjugates in high-throughput sequencing

[0148] The prepared antibody fragment and enzyme conjugate was subjected to sequencing test (the specific operation steps are described in Example 1), and the results showed that compared with the control group (full-length antibody-luciferase conjugate), the prepared small protein (i.e., antibody fragment-luciferase conjugate) ensured and improved the sequencing quality; during the entire sequencing process, the experimental group small protein (i.e., antibody fragment-luciferase conjugate) can significantly reduce the Runon / Lag during the entire sequencing process. Low Lag and low Runon can make the sequencing read length longer and the sequencing quality higher.

[0149] Example 3: Preparation of small molecular weight antibody fragment-enzyme conjugates by fusion expression method and application of the prepared antibody fragment-enzyme conjugates in high-throughput sequencing

[0150] 1. Preparation of antibody fragment (Fab / F(ab')2) and enzyme conjugates by fusion expression method

[0151] Fig.15 Exemplary structures of different antibody fragments expressed by fusion with luciferase.

[0152] We constructed a plasmid containing a Fab / F(ab') fragment that specifically recognizes digoxin, an optional linker, and Nanoluc luciferase on the same vector, wherein the quantitative ratio (or molar ratio) of Fab and Nanoluc luciferase molecules was controlled to be 1:1 (i.e., one Fab corresponds to one Nanoluc luciferase molecule); the quantitative ratio of F(ab')2 and Nanoluc luciferase was controlled to be 1:1 (i.e., one F(ab')2 corresponds to one Nanoluc luciferase molecule) and 1:2 (i.e., one F(ab')2 corresponds to one Nanoluc luciferase molecule). b')2 corresponds to two Nanoluc luciferase molecules); through eukaryotic expression, we obtained three expressions, among which the molecular weight of Fab-Nanoluc is about 65kDa, the molecular weight of F(ab')2-Nanoluc is about 130kDa, and the molecular weight of F(ab')2-2Nanoluc is about 150kDa; we compared the catalytic substrate luminescence ability and antigen-antibody affinity of the three expression products with the full-length antibody-Nanoluc conjugate (control group) by microplate reader and SPR. At 1μg / mL, the catalytic substrate luminescence ability of the three products (Fab-Nanoluc, F(ab')2-Nanoluc and F(ab')2-2Nanoluc) expressed by fusion is about 3, 3, and 6 times that of the control group (full-length antibody-Nanoluc conjugate), and the affinity is about 2 times.

[0153] II. Application of antibody fragment (Fab / F(ab')2) and enzyme conjugates in high-throughput sequencing

[0154] F(ab')2-2Nanoluc was selected as the experimental group, and its performance in the online results was tested by concentration titration. It was found that the F(ab')2-2Nanoluc conjugate at a working concentration of 0.5μg / mL could achieve the same signal intensity as the control group (full-length antibody-Nanoluc conjugate) at a working concentration of 3μg / mL, and had higher sequencing quality, lower Lag / Runon and base error rate.

[0155] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A high-throughput sequencing method, characterized in that: The method comprises the following steps: in a DNA amplification reaction of high-throughput sequencing, the first of the four dNTPs is labeled with substance 1, the second is labeled with substance 2, the third is labeled with the substance 1 and the substance 2, and the fourth is not labeled; then, substance 1'-luciferase 1 conjugate and substance 2'-luciferase 2 conjugate are used to distinguish the four dNTPs; The substance 1' can specifically bind to the substance 1; the substance 2' can specifically bind to the substance 2; at least one of the substance 1 and the substance 2 is an antigen or a hapten molecule, and correspondingly at least one of the substance 1' and the substance 2' is an antibody fragment that can specifically recognize the antigen or hapten molecule.

2. The method according to claim 1, characterized in that: A portion of the third of the four dNTPs is labeled with the substance 1, and another portion is labeled with the substance 2.

3. The method according to claim 1 or 2, characterized in that: The conjugate formed by the antibody fragment and luciferase is obtained by any of the following methods: (A1) cutting the full-length antibody that specifically recognizes the antigen or hapten molecule into the antibody fragment, and then connecting the antibody fragment to luciferase by chemical coupling; (A2) directly constructing a fusion plasmid for expressing the antibody fragment and luciferase conjugate by biosynthesis, and then introducing the fusion plasmid into a recipient cell or a recipient bacterium for expression; (A3) The antibody fragment is biosynthesized, and then the antibody fragment is linked to luciferase by chemical coupling.

4. The method according to claim 3, characterized in that: In (A1) and (A3), when the chemical coupling is performed, the antibody fragment exposes the sulfhydryl group by the action of a reducing agent or a non-reducing agent, the luciferase is activated by a coupling agent, and the treated antibody fragment and the activated luciferase are connected with or without the addition of a linker; Preferably, the antibody fragment exposes the sulfhydryl group by the action of a reducing agent, wherein the reducing agent is selected from dithiothreitol, tris(2-carbonylethyl)phosphine hydrochloride and mercaptoethylamine; Preferably, the coupling agent is selected from 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt, 2-iminothiolane hydrochloride; Preferably, the linker is bismaleimide or maleimide-polyethylene glycol-maleimide; More preferably, the reducing agent is tris(2-carbonylethyl)phosphine hydrochloride; More preferably, the coupling agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt; More preferably, the treated antibody fragment and the activated luciferase are linked without adding a linker.

5. The method according to claim 1 or 2, characterized in that: The antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, and a F(ab')2 fragment; Preferably, the antibody fragment is a Fab fragment or a F(ab')2 fragment; Preferably, the luciferase 1 and the luciferase 2 are respectively selected from firefly luciferase, bacterial luciferase, Renilla luciferase, Gaussia luciferase, Nanoluc luciferase and spiny shrimp luciferase; More preferably, the luciferase 1 is Gaussia luciferase, and the luciferase 2 is Nanoluc luciferase; More preferably, the substance 1 is biotin, the substance 1' is streptavidin; the substance 2 is digoxigenin, and the substance 2' is a Fab fragment or a F(ab')2 fragment that specifically recognizes digoxigenin.

6. Use of the method according to any one of claims 1 to 5 in any of the following: (B1) Improving the accuracy of sequencing data from high-throughput sequencing; (B2) Reducing Lag and / or Runon during high-throughput sequencing; (B3) Reduce the base mismatch rate during high-throughput sequencing.

7. Use of the method according to any one of claims 1 to 5 in preparing products of high-throughput sequencing technology.

8. A complete set of products for high-throughput sequencing or the use of the complete set of products in high-throughput sequencing, characterized in that: The complete set of products includes the following (C1)-(C3): (C1) 4 dNTPs according to any one of claims 1 to 5; (C2) a substance 1′-luciferase 1 conjugate according to any one of claims 1 to 5; (C3) The substance 2'-luciferase 2 conjugate according to any one of claims 1 to 5.

9. The method, application or set of products according to any one of claims 1 to 8, characterized in that: The high-throughput sequencing is second-generation sequencing.

10. A method for preparing an antibody fragment-luciferase conjugate or an antibody fragment-luciferase conjugate prepared by the method, characterized in that: The method comprises any of the following steps: (A1) cutting a full-length antibody that specifically recognizes an antigen or hapten molecule into the antibody fragment, and then connecting the antibody fragment to luciferase by chemical coupling; (A2) directly constructing a fusion plasmid for expressing the antibody fragment and luciferase conjugate by biosynthesis, and then introducing the fusion plasmid into a recipient cell or a recipient bacterium for expression; (A3) biosynthesizing the antibody fragment, and then connecting the antibody fragment to luciferase by chemical coupling; Furthermore, in (A1) and (A3), when the chemical coupling is performed, the antibody fragment exposes the sulfhydryl group by the action of a reducing agent or a non-reducing agent, the luciferase is activated by a coupling agent, and the treated antibody fragment and the activated luciferase are connected with or without the addition of a linker; Preferably, the antibody fragment exposes the sulfhydryl group by the action of a reducing agent, wherein the reducing agent is selected from dithiothreitol, tris(2-carbonylethyl)phosphine hydrochloride and mercaptoethylamine; Preferably, the coupling agent is selected from 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt, 2-iminothiolane hydrochloride; Preferably, the linker is bismaleimide or maleimide-polyethylene glycol-maleimide; More preferably, the reducing agent is tris(2-carbonylethyl)phosphine hydrochloride; More preferably, the coupling agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt; More preferably, the treated antibody fragment and the activated luciferase are linked without adding a linker; Preferably, the antibody fragment is selected from a Fab fragment, a Fab' fragment, and a F(ab')2 fragment; more preferably, the antibody fragment is a Fab fragment or a F(ab')2 fragment; more preferably, the antibody fragment is a Fab fragment or a F(ab')2 fragment that specifically recognizes digoxin; Preferably, the luciferase is selected from the group consisting of firefly luciferase, bacterial luciferase, Renilla luciferase, Gaussia luciferase, Nanoluc luciferase and spiny shrimp luciferase; more preferably, the luciferase is Nanoluc luciferase.

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