WO2005007851A1

WO2005007851A1 - The dna-antibody and the uses thereof

Info

Publication number: WO2005007851A1
Application number: PCT/CN2003/000715
Authority: WO
Inventors: Jian Wen
Original assignee: Liu, Hui
Priority date: 2003-07-18
Filing date: 2003-08-25
Publication date: 2005-01-27
Also published as: AU2003261574A1; CN1214118C; CN1482258A

Abstract

The present invention relates to a DNA-antibody. The antibody was prepared by the following steps: 1) constructing three engineering antibody libraries of DNA polymorphism; 2) screening the above library of DNA-antibody; 3) labeling and amplifying the DNA-antibody. The present invention also relates to use the DNA molecule having antigen specially binding activity as the antibody so as to outcome the deficiency of antibody due to the variability of antigen and a series of difficulties of developing, screening, preparing, purifying protein, and expensive cost and long-term of preservation.

Description

DNA antibodies and their applications

TECHNICAL FIELD

The present invention relates to an antibody, and in particular, to a technology using DNA as an antibody. Prior art prior to the present invention

The key to immunological technology is to prepare antibodies that bind to specific antigens with high specificity, high affinity and high titer. Since each antigen has several epitopes, the antibodies produced by the animal it immunizes are multiple antibodies raised against multiple epitopes, called polyclonal antibodies. Monoclonal antibodies are produced on the basis of animal immunization. Hybridoma cells formed by fusion of a single antibody-producing cell of the immunized animal with myeloma cells are produced asexually and are directed against a single epitope. Therefore, its immunoglobulin belongs to the same category, with a pure texture, so it has strong specificity and consistent affinity. Polyclonal antibodies eliminate most of the later work in the production of monoclonal antibodies. Polyclonal antibodies are obtained by directly taking serum for purification after immunizing animals. Animals are time-consuming and expensive. Both specificity and affinity are much worse than monoclonal antibodies, so multiple antibodies are gradually replaced by monoclonal antibodies.

The main advantage of hybridoma technology lies in its two basic characteristics. First, the monoclonal antibody produced by isolated clones is a very clear chemical, rather than a species that can be immunized with each animal or even the same animal. An ambiguous, non-homogeneous mixture that changes with different blood collections. Permanent culture can provide monoclonal antibodies with identical chemical structures without restriction. Second, hybridoma technology is an ideal method for preparing pure antibodies with impure antigens.

The achievements of hybridoma-monoclonal antibody technology and DNA recombination technology have promoted the development of genetically engineered antibody technology. The first genetically engineered antibodies appeared were human and mouse chimeric antibodies reported in the early 1980s, followed by humans. Reshaped antibody antibodies), small molecule antibodies, antibody fusion proteins, and other monoclonal antibodies that have been modified at the genetic level. From the late 1980s to the early 1990s, (1) direct expression of functional antibody molecule fragments in E. coli: (2) polymerase chain reaction (PCR) amplification of antibody variable region genes and ( 3) An antibody library technology has been established based on the development of efficient screening and other technologies through phage display systems, which can be used to clone genes of specific antibodies directly in prokaryotic cell systems by genetic engineering and produce the specific antibodies. This revolutionary development has had a profound impact on the preparation, transformation and development of new antibodies, marking that the antibody technology has gone through the first generation of antibodies, serum polyclonal antibodies, and the second generation of antibodies, monoclonal antibodies. Developed to the third generation of antibodies-genetically engineered antibodies.

Genetically engineered antibody technology consists of two parts;-Reconstruction of existing monoclonal antibodies using DNA recombination technology, including humanization of murine monoclonal antibodies, preparation of small molecule antibodies and antibody fusion proteins. The second is to use antibody library technology to screen and clone new monoclonal antibodies. Because genetic engineering antibody technology is still in the development stage. It is more exploratory, and many technologies do not have mature and standardized operating procedures. Different laboratories often adopt different technical methods.

The true genetic engineering of antibodies began in the end of 1989 in the creative work of the winter group in Cambridge, England and the lerner group in the Scrips Institute. They used the PCR method to clone all the antibody genes of the body and recombined them into the prokaryotic expression vector. The corresponding antibodies were screened by labeling the antigen, which was then called the combined antibody library technology. In the early 1990s, this technology has been further developed, that is, the antibody gene (vH or Fd) is fused with the coat protein of single-chain phage and expressed on the surface of the phage, and the corresponding phage antibody is adsorbed by the solid-phase antigen. After several "adsorption-elution-amplification", the desired antibody can be obtained. This is another major technological revolution in antibody production. First, the technology closely linked the genotype and phenotype of the antibody. Expressed phage antibody (also secreted) available for function Detection, amplification of phage and extraction of DNA can obtain corresponding antibody genes, which is convenient for sequencing or further genetic manipulation. Secondly, the technology has large capacity and high efficiency. Generally, the database can reach 10 ⁸ , which basically contains the information of antibody diversity. Third, "adsorption - elution - amplification" re-amplified with the selection process organically combine antibodies, specific antibodies can each run enriched ^{^102--103} times. The phage antibody library technology not only gets rid of complicated operations such as cell fusion, but also prepares antibodies without immunizing agents, opening a new way for preparing human-derived antibodies, which has been confirmed by experiments.

The antibodies used above have a common feature, that is, they are proteins, whether they are polyclonal antibodies, monoclonal antibodies or genetically engineered antibodies, the basic principles are the same: the use of heavy and light chain variable regions and epitopes For relative specific binding, a variety of specific antibodies can be produced by screening and enriching specific antibody-producing cells in mice or constructing an engineered antibody library in vitro and efficiently screening clones that produce specific antibodies.

However, the nature of polyclonal antibodies, monoclonal antibodies, and genetically engineered antibodies as proteins determines their inherent shortcomings, because various operations at the protein level such as expression, isolation, purification, and diagnosis require long cycles, complicated procedures, and Higher costs, the following uses monoclonal antibodies and genetically engineered antibodies to illustrate:

1. Long cycle: The immune cycle of animals in the production of monoclonal antibodies is at least one month, and fusion and screening identification plus cell cloning needs at least one month, plus ascites preparation and antigen antibody purification, etc., if it can be prepared within three months A sufficiently high specific and sensitive monoclonal antibody is already going well. Although the cycle of genetically engineered antibodies is shorter than that of monoclonal antibodies, it also takes more than one month, and the premise is that a sufficiently diverse (> 10 ⁹ ) engineering antibody library has been successfully constructed, and only specific antigens need to be used to screen for specific antibodies. The construction of antibody libraries is complicated, at least one month. However, the specificity and affinity of genetically engineered antibodies are not as good as monoclonal antibodies. Lengthy procedures and high preparation costs: The required operations in the preparation of monoclonal antibodies are lengthy and numerous, such as the preparation and purification of antigens in animal immunization, animal room preparation and animal culture, and certain blood samples are taken regularly to test the serum antibody titer; Preparation of myeloma cells, fusion and selection of cells, cloning of hybrid cells, selection and maintenance of positive clones, identification of specificity and titer, preparation of ascites, and purification of antibodies, etc. How many operations are required for preparation, and the reagents required for cell culture and protein purification are expensive, and the time is naturally high. Genetically engineered antibody antibody libraries are complicated to construct, and it is indeed a difficult problem to establish a diverse library (> 10 ⁹ ) that can truly represent the billions of diverse antibodies in the spleen cells; A few specific antibodies were screened out of the antibodies, and they also required high specificity and affinity, and purification of antigen antibodies, all of which still made them expensive and cumbersome to operate.

Other shortcomings of monoclonal antibodies:

] It is difficult to obtain specific antibodies or hybridoma cells for poorly immunogenic antigens. · The only way to deal with poorly immunogenic antigens is to increase the number of useful antibody-forming cells. Immunization protocols can be improved or antigens can be modified to increase their immunogens. However, these methods are quite complicated, because it is not completely clear whether antibody-forming cells or their precursors can form good hybrid cells. The available data suggest that the only way to overcome weak immunogenicity is to combine technologies, that is, to have sufficient suitable B cells were then fused with myeloma parent cells. The researchers used this method to concentrate the number of B cells 5-10 times, which has the advantage of reducing the amount of screening. However, such improvements are not enough to achieve the conventional acquisition of anti-weak immunogen hybridomas. The practical value of concentration technology needs to be further studied.

) Production of monoclonal antibodies other than mice and rats, especially domestic and human monoclonal antibodies Cause of difficulty.

3] At present, there are no successful humanized monoclonal antibodies, so it is not easy to use in medicine.

Deficiencies in genetically engineered antibody technology:

1) High requirements for the diversity of antibody libraries (> 10 ⁹ ), complicated construction and high construction cost.

2) As a protein antibody, there are still a series of problems in the production and purification, as well as the immunogenicity of protein molecules, and the expression of genetically engineered antibodies in prokaryotic cells mimics the structure and function of the parent antibody. After all, it is not as good as the affinity and affinity of the parent antibody. Specificity.

3) Screening is difficult. It is definitely not an easy task to select ideal antibodies from an unimmunized primary antibody library, and ideal antibodies cannot be screened for many antigens.

4) In the application of target drug screening, although the humanized engineered antibody can avoid its immunogenicity, the purity of the antibody is very high, and the humanized engineered antibody library is not easy to construct and screen. Object of the invention

It can be seen that the inherent contradiction of antibodies as proteins cannot be solved. The present invention creatively proposes the concept of DM antibodies, that is, the use of DNA molecules with specific antigen binding activity as antibodies, mainly to solve the problem of protein diagnosis due to antigens. The ever-changing variety always lacks the corresponding antibodies, and it is a series of problems such as high cost and long cycle caused by the difficulty of R & D, screening, production, purification and storage of antibodies as proteins.

Technical solution of the present invention

In the study of the interaction between DNA and protein, it was found that specific binding and interaction between DNA and protein and other macromolecules generally existed. The concept of DNA antibody was creatively proposed to solve the inherent problems existing in protein antibody. Three DNA antibody library constructions and their efficient screening methods have been created to ultimately obtain DNA antibodies. The above DNA antibody is prepared according to the following steps:

1) Construction of three kinds of DNA diversified engineering antibody libraries;

2) screening of DNA antibody library;

3) DNA antibody labeling and amplification.

The invention creatively proposes the concept of a DNA antibody, that is, using a DNA molecule with specific binding antigen activity as an antibody, mainly to solve the inherent defects of the previous protein antibodies, and due to the diversity of antigens, there is always a lack of corresponding antibodies and antibodies as proteins. It is not easy to develop, screen, produce, purify and preserve a series of problems such as high costs and long cycles, because in proteomics and gene function research and various immunological scientific research practices, many kinds of antigens are often produced The corresponding specific antibodies need to be obtained temporarily, and the production cycle of both monoclonal antibodies and genetically engineered antibodies is quite long, the production and purification processes are numerous, and the cost is high, which greatly limits the progress and quality of scientific research and development. In this sense, The production cycle of DNA antibodies is short and the cost is low. After they are produced, customers can even produce their own amplification, which will greatly promote the development of genomics and proteomics in the world today.

And because protein and antibody have too large molecular weight and strong immunogenicity, they are not easy to enter cells and are not easy to be used for target drugs; while DNA antibody molecules are small, have low immunogenicity and are easy to penetrate tissues, which just solves these shortcomings.

Moreover, under the premise of ensuring certain specificity and affinity of DNA antibodies, its various antibody libraries are very easy to construct, and its screening is simple and easy, because the screening is enriched by binding one by one and PCR. The process is carried out with a short cycle, and the preservation and replication of DNA antibodies is simpler and the cost is relatively low. In addition, it is easy to label and has small molecules. After further optimization, its specificity and sensitivity can be improved. Small molecules can also avoid immunogenicity, becoming antibodies and Preferred target drug for nucleic acid drugs.

The main thing is that, through the use of DNA antibodies, the diagnosis and analysis of proteins can be transferred to the diagnosis and analysis of DNA, thus bringing a new technology platform for proteomics. For example, transfer subtractive hybridization at the protein level to subtractive hybridization at the gene level. It can be said that DNA antibodies will open up a whole new situation for the diagnosis of proteins, and its application significance and prospects cannot be predicted now.

Comparison of the advantages and disadvantages of the three antibodies.- Types of antibodies Monoclonal antibodies Genetically engineered antibodies DNA antibodies Antibody properties Immunoglobulins Prokaryotically expressed polypeptides and protein molecules with a single, conformationally labeled chain DNA, double-stranded

DNA, hybrid double-stranded DNA

Preparation and in vivo screening enriched antibodies produced by constructing small storage capacity of the cell capacity by constructing method, by in vitro genetically engineered antibody with a myeloma library ^108, the amount exceeding ⁴ 4 ° over the cell fusion include screening for specific antibody diversity substantially DNA antibody library, hybridoma clone and identification. The correct information is based on three different types of Phage: the weak immunogen of the company can not be screened in the display method, which is far more than the specific monoclonal antibody; it is difficult to get high-efficiency screening. Insufficient library capacity has passed this number; hybridomas of species other than mice are often monoclonal antibodies enriched by genetically engineered antibodies. Reasons for complicated operation and failure of periodic screening. The operation method can efficiently screen long, high cost <= complicated, long cycle and high cost. Choose specific antibodies. Simple operation, short cycle and low cost.

Production and purification method Ascites or culture antibodies of hybridoma mice can be derived from prokaryotic expression of DNA antibodies. The supernatant can be obtained at any time and needs further purification. Cloning requires purification and removal of other preparations by PCR, which requires complicated operations and expensive proteins. The operation is complicated and gel purification is sufficient. High and low titer. The cost is high and the titer is not bad. Simple operation, low cost and extremely high titer. Preparation cost High preparation cost High preparation cost Low preparation cost At least 3 months At least 2 months One week

Diversity capacity can generate specific antibodies for most antigens because antibody libraries require sufficient DNA antibody libraries. However, it is simple and easy to generate a library of diverse antibodies with many immunizations. Low-genicity antigens. Because of hybridization, general antibody libraries can reach the limit of the efficiency of cross-tumor formation of diverse sequences. 10 ⁸ 'But due to the diversity of antigens Can be far away It is difficult to screen for its specificity, and still many anti-natural antibodies cannot be obtained. The amount of antigen-specific engineered antigen is sufficient. Specific engineered antibodies were obtained for most antigens.

Immunogenicity Large molecular weight, strong immunogenicity. Except for small molecule engineering antibodies, DNA antibodies are generally highly immunogenic small molecules with weak immunogenicity.

Specificity High specificity for antigen High specificity for antigen High specificity for antigen

Affinity Higher affinity for antigen Anti-affinity binding for antigen Not affinity for antigen, e.g., parent antibody is not as high as parent antibody

Titer Lower Lower High

Detection sensitivity Low Low High

Tissue Penetration Except for small molecule antibodies, other molecules are small and penetrating

Poor genetically engineered antibody tissue penetration good

Poor sex

For target drugs Monoclonal antibodies are difficult to use for target drugs, because humanized engineered antibodies can be used for target drugs and are highly immunogenic target drugs. However, the production is complicated and the production is easy.

It is used for chips. The number of arrays per chip is the number of arrays per chip. The number of arrays per chip is limited, and it is not easy to save. The limit of repetition is not easy to save. The number of repetitions is high. It is easy to maintain.

Examples

First, the basic principles of DNA antibody technology:

The production of monoclonal antibodies against various nucleic acids, especially DNA, implies that for single- or double-stranded DNA of different conformations and sequences, there is a specific binding antibody polymorphic region or antigen-binding site with different conformations and sequences This, in turn, means that for the antigenic epitopes of various proteins, there should be a single- or double-stranded DNA sequence that can specifically bind to it. Extensive research on the interaction of nucleic acids and proteins shows that there are a variety of proteins and enzymes that specifically interact with DNA in the body, and the specific regulation of proteins and enzymes by RNA all shows the nucleic acid sequence Specific interactions between columns and proteins are ubiquitous. In general, the specific recognition of DNA and protein lies in the combined effects of hydrogen bonding of bases and amino acids, electrostatic charge forces, anastomosis between sequence bone architecture images, van der Waals forces, and the like. Studies have shown that amino acid mutations in protein DNA binding sites can reduce this specific binding by three orders of magnitude.

DNA antibody technology basically consists of three parts: the construction of three kinds of DNA diversified engineering antibody libraries: screening of DNA antibody libraries; labeling and amplification of DNA antibodies.

Second, the specific technical route of DNA antibodies:

1) Construction of three diverse random DNA sequence engineering antibody libraries (double-stranded DNA random sequence library, single-stranded DNA random sequence library, hybrid double-stranded DNA random sequence library):

Diversification of the DNA sequence library is a prerequisite for efficient screening of specific antibodies, so the construction of its DNA antibody library is very important. By constructing a double-stranded DNA random sequence library, a single-stranded DNA random sequence library, and a hybrid double-stranded DNA random sequence library, the purpose of constructing a sufficient amount of diverse random DNA sequences is achieved. For heterocyclic double-stranded DNA, the stem-loop structure can stabilize the molecular structure and expose variable sequences, thereby increasing affinity and antibody stability. Multivalent antibodies have more affinity than monovalent antibodies. First, multiple monovalent antibodies are screened out, and then a sequence is linked to form a bivalent or multivalent antibody. The two monovalent antibodies are more flexibly linked together. Considering its small molecular volume, the conformation of the antigen may have little effect on the monovalent antibody, but it should have an effect on the bivalent antibody.

Design a random sequence 5 '-ggggggggggggggatccaac-N59-CTGCAGGTCGACGCAT-3' and a specific bow I (Fl) ggggggggggggatatacac and (Rl) atgcgtcgacctgcag for cloning at both ends for cloning; PCR amplification and cloning to a T vector library can be used to construct a random sequence representative library To save for future use.

Amplify 12 cycles with primers F1 and R1 at both ends, (94 ° C, 15 s; 55 ° C, 15 s; 72. C, 15 s) to obtain a double-stranded DNA random sequence library;

Using the double-stranded DM random sequence library as a template, and using one of the primers such as Fl primer for uneven horizontal PCR amplification for 45 cycles, (94 ° C, 15 s; 55 ° C, 15 s; 72. C, 15 s), The single-stranded DNA random sequence library can be obtained through 8% PAGE gel separation and purification;

Using this single-stranded DNA random sequence library as a template, add oligo (dC) at its 3 terminus, and put it at 75 degrees. 0 minutes to remove the single-stranded DNA conformation, inactivate the terminal transferase, and re-anneal at 4 degrees to form a hybrid The double-stranded DNA can be purified by 8% PAGE gel to obtain hybrid double-stranded random DNA sequence and single-stranded random DNA sequence library.

In order to facilitate the efficient enrichment of hybrid double-stranded DNA with specific binding antigen activity during the screening process, another scheme for constructing a hybrid double-stranded DNA random sequence library can also be adopted, that is, self-folding on the same single strand of DNA Formation of a handle-loop hybrid double-stranded structure: Design a random sequence 5, -ggggggggggggggggggggatccaac-N50-cccccccccccccggggggggggggggg ― N50-CTGCAGGTCGACGCAT-3 ', where the cccccccccccccccggggggggggggggg sequence is conducive to the formation of the hybrid double-stranded conformation, that is, the two sides are connected by this sequence The DNA sequence is folded. The hybrid double-stranded bands were also purified by PCR enrichment, followed by PCR and running gels.

2) Screening of a diverse random DNA sequence antibody library:

Preparation of specific antigen: The purified antigen was coated in 15 microwell plates (for protein) in a solution of (100 mM PIPES pH 6. 9, 1 mM EGTA, 0.5 mM MgS0; 100 uL) Coated, purchased from Corning Company] overnight, and 15 microwell plates were used to coat BSA or skimmed milk powder for pre-hybridization, for a total of 15 rounds of screening, about 1-10ug per well. PBS [including 0.1% Tween-20) was used to wash away excess antigen, and the microtiter plate was blocked with 3% BSA or 5% skimmed milk powder in PBS for 30 minutes, washed three times with PBS (containing 0.1% Tween-20), and Screening buffer (100 raM PIPES H 6. 9, 1 mM EGTA, 5 mM MgS04, 100 mM NaCl).

Pre-hybridization: 10ug of the three DNA random sequence libraries prepared above were pre-hybridized in a microplate coated with BSA or skimmed milk powder and combined in a screening buffer for 30 minutes.

Hybridization binding: Take the pre-hybridized DNA random sequence, transfer to a microtiter plate coated with specific antigen, and hybridize in screening buffer at 4 ° C for 30 minutes.

Washing: The non-specific unbound random DNA sequence was washed out with the screening buffer and washed three times for 30 minutes each.

Note: For different antigens, the best combination hybridization and washing conditions should be explored.

3) Elution and PCR enrichment of specific DNA antibodies:

DNA was extracted with an equal volume of phenol, and the supernatant [DNA part] was extracted after centrifugation. The DNA was precipitated with ethanol and sodium acetate, and the DNA was used as a template. The double-stranded DNA was first amplified by PCR with F1 and R1 primers. It is template uneven PCR amplification of single-stranded DNA sequence. Using this single-stranded DNA sequence as a template, add oligo-dC to its end, remove conformation at 75 degrees, and renaturate at 4 degrees. Double-stranded DNA, single-stranded DNA sequence, hybrid double-stranded DNA sequence. The PCR is the same as the uneven PCR reaction system.

4) Repeat the pre-hybridization-hybridization-washing-elution-PCR enrichment process for 15 rounds as above, and finally get three DNA sequences that specifically bind to the antigen (single-strand D-dish sequence, double-stranded DNA, hybrid double-stranded DNA Sequence).

5] Sequencing, storage, and amplification of specific DNA antibodies: While constructing PCR-T clones, select 10 sequencing, select which clones have the most sequence, and directly send sequencing to select the highest peak as the DNA sequence, such as The peak height is obvious and the background is low, which means that the DNA antibody is more specific and has a higher affinity for other DNA random sequences. The resulting clone or DNA sequence can be used as a template to save It is used directly to prepare and label DNA antibodies.

Sometimes a mixture of several kinds of DNA antibodies is required, and they can form a conformation to bind to the antigen molecule efficiently and specifically. Therefore, the screened sequence is generally used as a template for direct amplification without the need to isolate individual sequences. The maintenance of the sequence mixture can use the primers bound to the solid phase to synthesize a fixed template, which can be used repeatedly without significantly changing the proportion of the original DNA random sequence. Of course, several simple sequences can be further screened and their final composition determined by sequencing.

6) Labeling and production of DNA antibodies: Use the specific DNA sequence selected above as a template to amplify the selected specific DNA sequence by PCR with labeled (such as digoxin, biotin, radioactive elements such as P32) primers. The corresponding three kinds of DNA antibodies were prepared by PAGE gel purification. If labeled with digoxin or biotin, amplifying the signal with a secondary antibody is also possible. Or use colloidal gold, radioactive elements or other direct color or fluorescent excitation labels (note that the molecules used for labeling should be as small as possible).

7) DNA antibody specific identification: Serum and similar antigens were selected as controls, and the degree of cross-reaction between the specific DNA antibody and specific antigens and non-specific antigens was compared. Methods can be ELISA, IFA and so on.

8) DNA antibody affinity identification: Affinity indicates how tightly the antibody binds to the antigen, expressed as an affinity constant. Methods can use ELISA or RIA competition binding test.

9) Identification of DNA antibody antigen binding site sequence: Photochemical cross-linking, proteolytic cleavage, and amino acid sequencing are used to determine the epitope sequence that specifically binds to the DNA antibody.

Mix the antigen and DNA antibody in TE solution (select different concentrations of NaCl), incubate on ice for 20 minutes, transfer it to silicate glass plate with 0.25ml, and irradiate it under a 15W sterilized ultraviolet lamp (distance 8cm) Minutes, UV irradiation can cause intermolecular cross-linking in specifically bound complexes, Therefore, the affinity and specificity of the DNA antibody can be enhanced. The final concentration was 10%, and the cross-linked complex of protein antigen and DNA antibody and the rest of the protein antigen were precipitated by centrifugation. The remaining unbound DNA was not precipitated. Wash once with 10% TCA, three times with pre-chilled acetone, and air dry. Add 0.2M of 8M urea to the precipitate, and dissolve the precipitate with an sonicator (10 seconds each time, maximum power, three times). After dissolution, continue to add NH4HC03 to a final concentration of 2M urea, 0.1MNH4HC03o press 1: 25 (pancreatin : Protein-antibody complex] with a mass ratio of trypsin, 37 "C, digestion for 24 hours. The digestion product is passed through an ion exchange column, and the most radioactive part (ie, the epitope peptide or amino acid sequence specifically bound by the DNA antibody) is removed. Sequencing (note that the DNA antibody is labeled with radioactive element P32).

10] Improved affinity and specificity of DNA antibodies and detection sensitivity:

a. By increasing the number of screening rounds, several kinds of antibodies with high specificity and affinity are screened from the three DNA random antibody libraries. Since each DNA antibody specifically recognizes different antigenic epitopes, they can be used in combination to improve Specificity. To increase the detection sensitivity of DNA antibodies, you can label the antibody with P32.

b. For the production of DNA antibodies for each antigen, an optimal binding and washing conditions must be given at the same time to increase the specificity and affinity of the DNA antibodies for specific antigens and reduce the non-specific background.

c. UV cross-linking and proteolysis can be used to determine whether the DNA antibody binding site is a specific non-conservative sequence of the antigen, and the amino acid sequence of the antigenic epitope to which it binds can be directly analyzed by sequencing.

d. A series of conditions can be changed to increase the concentration of the DNA antibody, thereby enhancing the affinity of the DNA antibody to the antigen, and the minimum detection concentration of the antigen should be reduced. e. Ultraviolet irradiation can be used to cross-link the specifically bound DNA antibody with the antigen. SDS and salt are used to wash away the non-specifically bound DNA antibody, and the in situ PCR enrichment with labeled primers is used to further develop the DNA antibody detection Sensitivity and specificity.

f. By modifying the monovalent antibody into a multivalent antibody, the affinity and specificity of the antibody should be multiplied. g. Rebuild the DNA antibody library, such as changing to another DNA antibody library, and increasing the length of the random sequence.

h. Stem-loop structure and multivalent DNA antibody can improve the affinity and specificity of its monovalent antibody. Hybrid double-stranded stem-loop structure can stabilize and appropriately adjust the conformation of the antigen-binding site in the DNA antibody, so as to better Specific binding with antigen; and by combining multiple monovalent DNA antibodies into multivalent antibodies, the affinity and specificity of DNA antibodies will be doubled.

i. For the hybrid double-stranded DNA sequence antibody library, the GC sequence in the middle can use the AT sequence.

Application:

The DNA antibody technology can be widely used in immunological fields such as immunoimprinting, immunohistochemistry, specific gene regulation at the protein level, protein tracking, antibody target drug screening, affinity chromatography, and protein subtractive hybridization.

In the fields of immunoimprinting, immunohistochemistry, specific gene regulation at the protein level, protein tracking, and antibody target drug screening, the use of the DNA antibody is basically similar to the use of ordinary antibodies. Due to the easy availability of DNA, the above applications have been greatly simplified, both in time and cost. Such as affinity chromatography, such as the use of protein antibodies as affinity columns, the price is obvious, and if you want to make your own affinity columns, from antibody preparation to affinity columns, this is a long and costly the process of.

And DNA antibodies can also be used in various fields like other antibodies: (1) Immunogenicity group Weave compatible antigens or differentiation antigens, (2) differentiation antigens, tumor antigens, or other cell surface antigens. These antigens lack polymorphism and are not immunogenic in the same system, but can be recognized in heterogeneous immunity. (3) virus and bacterial antigens; (4) a single epitope family of various proteins, nucleic acids and sugar surfaces. Such antibodies allow us to identify and isolate cell subgroups, distinguish between different stages of cell development, and perform more accurately Tissue typing, purification of small surface antigens, fine identification of microorganisms for diagnostic and epidemiological studies, and more reliable immunological analysis and diagnosis of various biological macromolecules.

Protein subtractive hybridization is a new method to systematically look for protein expression differences at the protein level. Because these proteins that have differential expression between pathological cells and normal cells must have a certain signal pathway or connection, it is an intracellular system. Various complex signaling pathways and macromolecular interactions provide true data directly at the protein level. Most of the differences in the expression of genes studied in the past are at the RNA level. Because the post-transcriptional modifications, translation levels, and post-translational changes have been ignored, they cannot truly reflect the final changes in protein levels. As for The research on the display of the difference in protein level, due to the difficulty of the operation of the protein, most of the time is to use the antibody chip method, can only find the changes in the expression of known proteins, can not find new proteins, and the current cost of antibody chips is extremely high. High, its use requires precision instruments, ordinary scientific research institutes cannot afford it, and the amount of antibodies contained in antibody chips is limited, which is far less than the number of gene chips, and it cannot really achieve the purpose of high-throughput screening. However, this method displays the difference in expression at the protein level and transfers the DNA antibody to the difference display at the DNA level, thereby quickly and easily achieving the purpose of an open high-throughput screening of differentially expressed proteins, which is a system of protein correlation. Research and systematic exploration of gene functions provide a shortcut, and the technology is generally applicable to large, medium and small scientific research institutes and companies. Protein subtractive hybridization:

The basic principle is to access specific antigen protein molecules through the DNA random antibody library into the cell. If a protein molecule has a different number of expressions, the specific DNA sequence that it binds will also show a corresponding number change. Subtractive hybridization at the protein level is transferred to subtractive hybridization to DNA sequences.

1) The above three kinds of DNA random sequence libraries are incubated with control cells and target cells (living cells) that have been fixed (the number of cells and other conditions are as consistent as possible). Under certain conditions, the DNA random sequence libraries will enter the cells and be specific. Binding to its corresponding molecule, and then washing away DNA antibodies that do not enter the cell sequence or non-specifically bind to cellular proteins.

2) Cells were lysed with phenol-chloroform and protein was extracted. The supernatant [containing the DNA sequence] DNA was collected, and the DNA was concentrated in a vacuum dryer or precipitated with alcohol to obtain a DNA random sequence library that can reflect changes in protein levels.

3) After performing subtractive hybridization (using a kit) on random DNA sequence libraries of target cells and control cells, note that a labeled internal control sequence is added to detect the subtractive hybridization effect before subtraction; PCR subtracts the sequence to obtain a differential display library and Sequencing, selecting several to dozens of clones, amplifying the corresponding types of DNA antibodies with labeled primers, and detecting their specificity with serum, removing the DNA sequence of non-specifically bound serum.

4) Use this DNA antibody to label and bind the corresponding protein molecules in the cell to form a complex. First, irradiate with UV for tens of minutes to form covalent cross-links, and then run SDS-PAGE gel to detect the label.

-Protein complex, bands were excised, purified and sequenced. Or if it is non-membrane protein, and the efficiency of forming a cross-linked complex by UV irradiation is low, it can be gently lysed, and the corresponding primer-cellulose affinity chromatography can be used to separate and purify the DNA-protein complex. It can also be separated and purified by PAGE gel. , Measurement Sequence to obtain the protein sequence.

5) In the case of membrane proteins, first cross-link DNA molecules and antigen molecules with ultraviolet radiation, run SDS-PAGE gel to separate and purify the labeled DNA-protein complex, cut out the band, and purify and sequence. Or use affinity chromatography as above.

V. Specific preparation and implementation examples:

Tubulin screens for specific DNA antibodies for antigens

1] Single-stranded DNA random sequence engineering antibody library was used to screen tubulin DNA antibodies: Design random sequence 5'-ggggggggggggatccaac-N59-CTGCAGGTCGACGCAT-3 'and specific primers (Fl) ggggggggggggggatccac and (Rl) atgcgtcgacctgcag at both ends For cloning; PCR amplification can be cloned into the T vector library to construct a random sequence representative library for storage. N59 refers to a random sequence of 59 nucleotides.

Amplify 12 cycles with primers F1 and R1 at both ends (94 ° C, 15 s; 55 ° C, 15 s; 72 ° C, 15 s), then you can get a double-stranded DNA random sequence library;

Using the double-stranded DNA random sequence library as a template, and using one of the primers such as F1 primer for uneven horizontal PCR amplification for 45 cycles, (94 ° C, 15 s; 55. C, 15 s; 72. C, 15 s), The single-stranded DNA random sequence library can be obtained through 8% PAGE gel separation and purification;

2] Screening of diverse random DNA sequence engineering antibody libraries:

The purified tubulin antigen was added to a solution of (100 mM PIPES pH 6.9, 1 mM EGTA, 0.5 mM MgS04; 100 uL) in 20 microwell plates (for protein coating, Purchased from Corning Company] overnight, and another 20 micro-well plates were used to coat BSA or skimmed milk powder for pre-hybridization, a total of 20 rounds of screening, about 1-10ug per well. PBS [containing 0.1 % Tween-20] Wash away excess antigen, block the microplate with 3% BSA or 5% skimmed milk powder in PBS for 30 minutes, and use PBS (Containing 0.1% Tween-20), three times, and once with screening buffer (100 mM PIPES pH 6. 9, 1 mM EGTA, 5 mM MgS04, 100 mM NaCl).

Pre-hybridization: Use the prepared single-stranded DNA random sequence library 10 ng above to pre-hybridize in a microplate coated with BSA or skimmed milk powder, and bind in screening buffer for 30 minutes.

Hybridization binding: Take the pre-hybridized DNA random sequence, transfer it to the microtiter plate coated with specific antigen, and hybridize in the screening buffer at 4 ° C for 30 minutes.

Washing: Non-specific unbound random DM sequences were washed out with screening buffer and washed three times for 30 minutes each.

3) Elution and PCR enrichment of specific DNA antibodies: DNA is extracted with an equal volume of phenol, and the supernatant (DNA part) is extracted after centrifugation. The DNA is precipitated with ethanol and sodium acetate, and the DNA is used as a template. F1 and R1 primers were used for template amplification to enrich double-stranded DNA, and then used it as a template to amplify single-stranded DNA sequences and run 8% PAGE gel purification.

4) Repeat the pre-hybridization-hybridization-washing-elution-PCR enrichment process for 20 rounds as above, and finally obtain the DNA sequence that specifically binds the antigen.

5) Sequencing of specific DNA antibodies:

Using the specific DNA antibody screened above as a template, F1 and R1 were used to amplify the PCR products to construct the T vector. Twenty clones were randomly selected and sequenced for homology comparison. The resulting clone or DNA sequence can be used as a template for preservation.

The following sequences were obtained by sequencing (without primer sequences)-

ATGCTCGCGCCTGCTGTGTTGTTGCTTGGTTTGGTCTTTTTTTGGTTTGTTTTGGGTT; GAATTCGTTTGTGTGCGGAGGTGGTTGTTTGTTTTTTTTTTTTTTTTTTTTTTGTTTG; AGATATGGTTTTGTTGTGCGTTATGTTGTGTTTTTGGGGTCTCTTTTTGGGTGTTTTGT;

TTGGTGGGTTGTAGGCAGGCGTGGGCACTGTTTGAGAAGGACGTGTTTGGTCTAT;

6] DNA antibody labeling and amplification: Use the specific DNA sequence selected above as a template, and use the labeled (such as digoxin, biotin, radioactive elements such as P32) primers to amplify the selected specific DNA by PCR. The sequence was purified by PAGE gel to prepare the corresponding DNA antibody.

7) Specific identification of DNA antibodies Affinity identification: Because tubulin is purified from the calf brain, calf serum and similar antigens such as actin and fibrin are selected as non-specific antigen controls, and the specific DNA antibody pair is compared. Specificity of specific antigen and non-specific antigen response; At the same time, the single-stranded DNA random sequence before screening is used as a control, and the specific binding of the specific DNA antibody sequence and the random DNA sequence before screening to tubulin are compared. Detection was performed by ELISA method. The results showed that the specific DNA antibodies obtained from the screening had significantly greater binding power to tubulin than the random sequence before screening, and significantly greater binding to non-specific antigens. At the same time, for the binding of non-specific antigens, there was no significant difference between the specific DNA antibody and the random sequence of DNA before screening.

8) Affinity identification: Affinity indicates the ability of an antibody to bind to an antigen, and is expressed by the inverse of the dissociation constant (KD) of a DNA antibody-antigen complex. KD is the molar concentration at which 50% of the maximum effect occurs (50% of the receptors are occupied). The above DNA antibody dissociation constant is 20-45uM. By increasing the DNA sequence antibody concentration, the affinity can be improved.

Claims

1. A DNA antibody, characterized in that it is prepared according to the following steps:

1) Construction of DNA diversified antibody library;

2) screening of DNA antibody library;

3) Labeling and amplification of specific DNA antibodies.

2. The DNA antibody according to claim 1, wherein the construction of the DNA diversified antibody library includes the construction of a double-stranded DNA random sequence library, a single-stranded DNA random sequence library, and a hybrid double-stranded DNA random sequence library. .

3. The DNA antibody according to claim 1, characterized in that: screening of the DNA antibody library comprises specifically hybridizing and binding the random DNA sequence of the DNA antibody library and the antigen, and eluting the non-specifically bound random DNA sequence, and Elution and PCR enrichment of specific DNA antibodies.