CN116355959A - Expression vector of recombinant antibody and preparation method of recombinant antibody of interest in expression - Google Patents
Expression vector of recombinant antibody and preparation method of recombinant antibody of interest in expression Download PDFInfo
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Abstract
The invention discloses an expression vector of a recombinant antibody, in particular an expression vector pDAI containing an expression element of a mutant hCMV promoter and a chimericIntron, WPRE, BGHpolyA terminator, and also discloses a preparation method of the expression vector of the recombinant antibody for expressing a recombinant antibody of interest. The invention belongs to the technical field of bioengineering, and provides an expression vector of a recombinant antibody and a preparation method of the recombinant antibody for expressing the recombinant antibody, which provide an expression vector pDAI of an expression element containing a mutant hCMV promoter, chimericIntron, WPRE and BGHpolyA terminator, and a method for preparing the expression vector of the recombinant antibody for expressing the recombinant antibody by adopting a P2A sequence connected with an antibody light chain and an antibody heavy chain.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to an expression vector for recombinant antibodies and a preparation method for expressing recombinant antibodies of interest.
Background
The traditional antibody technology mainly comprises rabbit polyclonal antibody technology and mouse hybridoma technology, and has the main defects that the preparation process depends on animals, so that the batch difference caused by individual differences of the animals cannot be overcome, and the forcible use of animals for preparing the antibody is also a trend along with the enhancement of the knowledge of human beings on animal protection. The new generation recombinant antibody technology has two obvious advantages compared with the traditional hybridoma technology, namely the defect that hybridoma cells are easy to lose can be fundamentally prevented, so that the immortalization of the antibody is truly achieved. In addition, animal dependence can be fundamentally eliminated, so that batch-to-batch differences caused by animal batch differences are prevented, the stability and consistency of the product are ensured, and the method is particularly suitable for large-dose monoclonal antibody industrialized use.
The basic structure of an intact antibody is two identical heavy chain polypeptides and two identical light chain polypeptides. The heavy and light chains are held together by disulfide bonds and non-covalent interactions. The chain itself comprises a variable region and a constant region, the variable regions of the heavy and light chains being located at the N-terminal portion of the antibody molecule, the amino acid sequence of which is more susceptible to change than the constant region. Recombinant antibodies are produced by a series of technical means such as purification after the heavy chain gene fragment and the light chain gene fragment in the antibodies are introduced into host cells by means of molecular biology to express and assemble proteins.
Similar to the high-yield expression of recombinant proteins, for example, designing an expression vector with an optimal expression cassette with a strong promoter, the final product yield can be improved by adopting an efficient mammalian expression system of high-density suspension cells-Chinese Hamster Ovary (CHO) cells, optimizing culture media, culture conditions and the like. The most important difference between recombinant antibodies and recombinant protein expression is that the ratio of light chain protein to heavy chain protein expressed by the recombinant antibodies in mammalian cells has a significant impact on the final yield of recombinant antibodies. Therefore, designing a high-yield recombinant antibody expression vector is a key element of recombinant antibody expression technology. At present, the conventional method comprises the steps that the heavy chain gene fragment and the light chain gene fragment of a recombinant antibody are respectively loaded on expression vectors of two independent mammal expression systems, and the complete antibody product is finally obtained through cotransfection of the two expression vectors; or the expression vector of the same mammal expression system adopts two independent expression cassettes of heavy chain protein and light chain protein, and the final product is obtained after the plasmid is transfected. On one hand, the methods increase the working difficulty of the construction and expression process of upstream molecules, and on the other hand, the RNA transcription levels of heavy chains and light chains are uncontrollable, so that the problems of unsuitable assembly proportion, reduced assembly proportion of functional recombinant antibodies and finally reduced recombinant antibody yield are caused. The single plasmid expression vector for expressing the recombinant antibody provided by the invention adopts one expression cassette to express the heavy chain and the light chain simultaneously, reduces the difficulty of molecular construction and cell transfection, and ensures the consistency of RNA transcription levels of the heavy chain and the light chain. Experimental results show that the vector is used for loading the published Rituximab, trastuzumab and Muromina b-CD3 antibody sequences, is simpler to construct than a conventional double-plasmid expression vector, and can effectively improve the expression yield of recombinant antibodies.
Disclosure of Invention
In order to overcome the defects, the invention provides an expression vector of a recombinant antibody and a preparation method of the recombinant antibody for expressing the interest.
The invention provides the following technical scheme: the invention provides an expression vector of a recombinant antibody, in particular to an expression vector pDAI containing expression elements of a mutant hCMV promoter, a chimeric Intron and a WPRE-BGH polyA terminator.
Meanwhile, the invention also provides a preparation method for expressing the recombinant antibody of interest by using the expression vector of the recombinant antibody, which specifically comprises the following steps:
(1) Construction of expression vector pDAI
Sequence optimization design is carried out on sequence information to chemically synthesize a gene sequence (hCMV (M) -chimeric Intron) containing a mutant hCMV promoter and a chimeric Intron, and the gene sequence (WPRE-BGH polyA) of a WPRE and a BGH polyA terminator is chemically synthesized and connected to a pCDNA3.1 vector (enzyme cutting site Mlu I/Bbs I) framework which retains replicon and resistance genes through a homologous recombination method;
primer sequence:
F1：5‘-CGGGCCAGATATACGCGTTACATTGATTATTGACTAGTTATT-3’
F2：5‘-tccacaggtgtccagCGGATCCGAATTCTCGAaatcaacctct-3’
R1：5‘-agaggttgattTCGAGAATTCGGATCCGctggacacctgtgga-3’
R2：5‘-ATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTC-3’
first round PCR amplification system (total system 50 ul):
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 10s,30 cycles, holding at 16 ℃; amplifying a p1 fragment containing hCMV (M) -chimeric Intron; under the same conditions, replacing the upstream and downstream primers with F2 and R2, and amplifying a p2 fragment containing WPRE-BGH polyA;
second round PCR amplification system (overall 50 ul):
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10s, annealing at 65 ℃ for 5s, extension at 72 ℃ for 20s,30 cycles, holding at 16 ℃;
cloning the second round pcr product to a pCDNA3.1 vector with an enzyme cleavage site of Mlu I/Bbs I to construct an expression vector pDAI, and performing gel running and sequencing verification to obtain a successful vector construction;
(2) Construction of expression vectors Rituximab-pDAI, trastuzumab-pDAI, murominab-CD 3-pDAI
Chemically synthesizing a gene sequence containing a Kozak sequence, an antibody light chain of interest, P2A and an antibody heavy chain of interest, connecting the gene sequence to an expression vector pDAI (cloning site BamHI/EcoRI) by an enzyme digestion enzyme ligation method, and performing gel running and sequencing verification to prove that the vector is successfully constructed;
(3) Recombinant antibody expression plasmid Rituximab-pDAI, trastuzumab-pDAI, murominab-CD 3-pDAI extraction and CHO cell transfection, and other expression plasmid transfection steps are similar
Extracting: inoculating 10ml of bacteria according to an operation manual by using a Life plasmid DNA extraction kit, taking 2 mu L of the extracted plasmid, measuring the concentration by using a nucleic acid tester (Nanodrop One), taking 3 mu L of gel running electrophoresis detection and verification, and preserving the extracted plasmid at-20 ℃;
CHO cell transfection:
the day before transfection, cell counts were taken and 0.5X106/ml X30 ml was transferred to a new 125ml shake flask. If the cells are aggregated, the cells need to be filtered by a cell screen before counting; on the day of transfection, 2ml centrifuge tubes were prepared and 0.8ml serum-free medium was added, respectively; one tube was filled with 30. Mu.g of plasmid and the other tube with 60. Mu.g PEI, gently tumbling the tube and mixing well several times; adding diluted PEI into diluted plasmids, slightly turning over a centrifuge tube, uniformly mixing for several times, and incubating for 15min at room temperature; slowly adding the mixture into 30ml of prepared cells, and shaking while adding to complete transfection; continuing to culture until the 6 th day for sample collection;
(4) Purification of the products of the expression plasmids Rituximab-pDAI, trastuzumab-pDAI, muromina b-CD3-pDAI and comparison of the expression levels with other double plasmid expression vectors
Centrifuging the transfected culture solution, filtering the supernatant with 0.22um membrane, dialyzing at 4deg.C to Na with concentration of 20mmol/mL and pH=7.0 containing 150mmol/mL NaCl 2 HPO 4 And (3) in the buffer solution, purifying by using a ProteinA column after the dialysis is finished, and obtaining the recombinant antibody protein.
Further, the nucleotide sequence of hCMV (M) -chimeric Intron is:
ACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGGGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCCAGCggtttagtgaaccgtcagatcctgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccag。
further, the nucleotide sequence of the WPRE-BGH polyA is as follows:
AatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgTCTAGACGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG。
further, the nucleotide sequence of Rituximab-pDAI is:
gccgccaccATGGTCCTCCAGACCCAGGTGTTCATCAGTCTGCTGCTGTGGATTAGCGGCGCCTACGGCCAGATCGTGCTGAGCCAGAGCCCCGCCATCCTGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATCCACTGGTTCCAGCAGAAGCCCGGCAGCAGCCCCAAGCCCTGGATCTACGCCACCAGCAACCTGGCCAGCGGCGTGCCCGTGCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCCGCGTGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGACCAGCAACCCCCCCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACCGCGGCGAGTGCGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGAAGCACCTGTGGTTCTTTCTGCTGCTCGTGGCTGCTCCCAGATGGGTCCTGAGCCAGGTGCAGCTGCAGCAGCCCGGCGCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACCCCCGGCCGCGGCCTGGAGTGGATCGGCGCCATCTACCCCGGCAACGGCGACACCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCAGCACCTACTACGGCGGCGACTGGTACTTCAACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCGCCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGCCGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGCTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG。
further, the nucleotide sequence of Trastuzumab-pDAI is:
gccgccaccATGGTCCTCCAGACCCAGGTGTTCATCAGTCTGCTGCTGTGGATTAGCGGCGCCTACGGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCCGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGCACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACCGCGGCGAGTGCGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGAAGCACCTGTGGTTCTTTCTGCTGCTCGTGGCTGCTCCCAGATGGGTCCTGAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGCATCTACCCCACCAACGGCTACACCCGCTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCAGCCGCTGGGGCGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGCGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGCTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG。
further, the nucleotide sequence of the Murominab-CD 3-pDAI is:
gccgccaccATGGTCCTCCAGACCCAGGTGTTCATCAGTCTGCTGCTGTGGATTAGCGGCGCCTACGGCCAGATCGTTCTGACACAGAGCCCCGCTATCATGAGCGCTTCCCCAGGCGAGAAGGTGACCATGACCTGTAGCGCCTCTAGCAGCGTGTCTTACATGAACTGGTACCAGCAAAAGAGCGGCACAAGCCCTAAAAGATGGATCTACGACACCTCTAAACTGGCCAGCGGAGTGCCTGCCCACTTCCGGGGCAGCGGCAGCGGCACCTCTTATAGCCTGACCATCAGCGGTATGGAAGCTGAAGATGCCGCCACCTACTACTGCCAGCAATGGTCCAGCAACCCTTTTACCTTCGGCAGCGGCACAAAGCTGGAAATCAATAGAGCCGATACAGCCCCTACCGTGTCCATCTTCCCTCCCAGCAGCGAGCAGCTGACATCTGGAGGCGCCAGCGTGGTGTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGATCGACGGCTCTGAAAGACAGAACGGCGTGCTGAATAGCTGGACAGATCAGGACAGCAAGGACTCTACATACAGCATGTCCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACCTGCGAGGCCACACACAAGACCAGCACCTCCCCTATCGTGAAATCTTTTAACAGAAACGAGTGCGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGAAGCACCTGTGGTTCTTTCTGCTGCTCGTGGCTGCTCCCAGATGGGTCCTGAGCCAGGTGCAGCTGCAACAAAGCGGCGCCGAGCTGGCTAGACCTGGCGCTTCTGTGAAGATGAGCTGTAAAGCCAGCGGCTATACCTTTACCAGGTATACCATGCACTGGGTGAAGCAGCGGCCTGGCCAGGGCCTGGAATGGATCGGCTACATCAACCCCAGCCGGGGCTACACAAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACTGATAAGAGCTCTTCTACAGCCTACATGCAGCTGTCTTCTCTGACAAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTATTGTCTGGATTACTGGGGCCAGGGCACCACCCTGACCGTGTCCAGCgctagcGCAAAGACGACCGCCCCCAGCGTGTACCCTCTGGCCCCTGTGTGCGGAGGCACCACAGGCAGCTCCGTGACACTGGGCTGTCTGGTGAAAGGCTACTTCCCTGAACCCGTGACCCTGACCTGGAACAGCGGTAGCCTGTCCTCCGGAGTTCATACATTCCCCGCCGTGCTGCAGAGCGATCTGTACACACTGAGCAGCTCTGTGACCGTGACCAGCAGCACATGGCCTAGCCAGAGCATTACATGCAACGTGGCCCACCCCGCCAGCAGCACCAAGGTGGACAAAAAGATCGAGCCCAGACCAAAGAGCTGCGACAAAACACACACCTGCCCTCCTTGCCCCGCCCCAGAACTGCTGGGGGGCCCTTCTGTGTTCCTGTTTCCACCTAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAGGTGACCTGCGTGGTGGTCGACGTGAGCCATGAGGACCCTGAGGTGAAATTCAACTGGTACGTGGACGGCGTGGAAGTCCACAACGCCAAGACAAAGCCCCGGGAAGAGCAGTACAATAGCACCTACAGAGTGGTGTCTGTCCTGACCGTTCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAGACAATCTCCAAAGCCAAGGGCCAGCCTAGAGAGCCCCAGGTGTACACCCTGCCTCCTTCCCGCGACGAGCTGACAAAGAACCAGGTGTCCCTCACCTGTCTGGTGAAAGGCTTCTACCCCAGCGACATCGCCGTGGAATGGGAGAGCAACGGACAACCTGAAAACAACTACAAGACCACCCCTCCAGTGCTGGACTCCGATGGCTCCTTCTTCCTCTACAGCAAGCTGACCGTGGATAAGTCTCGGTGGCAGCAGGGAAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGCCTGTCACCTGGAAAGTAAGAATTC。
the beneficial effects obtained by the invention by adopting the structure are as follows: the invention provides an expression vector of a recombinant antibody and a preparation method of the recombinant antibody for expressing the recombinant antibody, and provides an expression vector pDAI containing expression elements of mutant hCMV promoter, chimeric Intron, WPRE and BGHpolyA terminator, and a method for preparing the recombinant antibody expression vector of interest by adopting P2A sequences connected with an antibody light chain and an antibody heavy chain and using the pDAI; the construction process of the expression vector is simple and the expression quantity of the antibody can be effectively improved by constructing a recombinant antibody (Rituximab, trastuzumab, murominab-CD 3) expression vector; the preparation process is simple, the recombinant antibody can be stably and effectively expressed in mammalian cells, the universality is strong, and the expression yield of the recombinant antibody is high.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a diagram of a pDAI expression vector comprising a mutant hCMV promoter, chimeric Intron, WPRE, BGH polyA terminator of the present invention;
FIG. 2 is a schematic representation of the light and heavy chains, and the P2A sequence linking the light and heavy chains, of a recombinant antibody of interest comprising a pDAI expression element of the invention;
FIG. 3 shows the results of PCR amplified nucleotide electrophoresis of heavy and light chain genes comprising an antibody of interest (Rituximab, trastuzumab, muromina b-CD 3) of the present invention;
FIG. 4 is a diagram of purified SDS-PAGE electrophoresis of recombinant expression of a recombinant antibody of interest (Rituximab, trastuzumab, muromina b-CD 3) of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that the words "front", "rear", "left", "right", "upper" and "lower" used in the following description refer to directions in the drawings, and the words "inner" and "outer" refer to directions toward or away from, respectively, the geometric center of a particular component.
The invention provides an expression vector of a recombinant antibody, in particular an expression vector pDAI of Wie containing expression elements of a mutant hCMV promoter, a chimeric Intron and a WPRE-BGHpolyA terminator.
Meanwhile, the invention also provides a preparation method for expressing the recombinant antibody of interest by using the expression vector of the recombinant antibody, which specifically comprises the following steps:
(1) Construction of expression vector pDAI
Sequence optimization design is carried out on sequence information to chemically synthesize a gene sequence (hCMV (M) -chimeric Intron) containing a mutant hCMV promoter and a chimeric Intron, and the gene sequence (WPRE-BGH polyA) of a WPRE and a BGH polyA terminator is chemically synthesized and connected to a pCDNA3.1 vector (enzyme cutting site Mlu I/Bbs I) framework which retains replicon and resistance genes through a homologous recombination method;
primer sequence:
F1：5‘-CGGGCCAGATATACGCGTTACATTGATTATTGACTAGTTATT-3’
F2：5‘-tccacaggtgtccagCGGATCCGAATTCTCGAaatcaacctct-3’
R1：5‘-agaggttgattTCGAGAATTCGGATCCGctggacacctgtgga-3’
R2：5‘-ATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTC-3’
first round PCR amplification system (total system 50 ul):
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 10s,30 cycles, holding at 16 ℃; amplifying a p1 fragment containing hCMV (M) -chimeric Intron; under the same conditions, replacing the upstream and downstream primers with F2 and R2, and amplifying a p2 fragment containing WPRE-BGH polyA;
second round PCR amplification system (overall 50 ul):
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10s, annealing at 65 ℃ for 5s, extension at 72 ℃ for 20s,30 cycles, holding at 16 ℃;
cloning the second round pcr product to a pCDNA3.1 vector with an enzyme cleavage site of Mlu I/Bbs I to construct an expression vector pDAI, and performing gel running and sequencing verification to obtain a successful vector construction;
(2) Construction of expression vectors Rituximab-pDAI, trastuzumab-pDAI, murominab-CD 3-pDAI
Chemically synthesizing a gene sequence containing a Kozak sequence, an antibody light chain of interest, P2A and an antibody heavy chain of interest, connecting the gene sequence to an expression vector pDAI (cloning site BamHI/EcoRI) by an enzyme digestion enzyme ligation method, and performing gel running and sequencing verification to prove that the vector is successfully constructed;
(3) Recombinant antibody expression plasmid Rituximab-pDAI, trastuzumab-pDAI, murominab-CD 3-pDAI extraction and CHO cell transfection (other expression plasmid transfection procedures are similar)
Extracting: inoculating 10ml of bacteria according to an operation manual by using a Life plasmid DNA extraction kit, taking 2 mu L of the extracted plasmid, measuring the concentration by using a nucleic acid tester (Nanodrop One), taking 3 mu L of gel running electrophoresis detection and verification, and preserving the extracted plasmid at-20 ℃;
CHO cell transfection:
the day before transfection, cell counts were taken and 0.5X106/ml X30 ml was transferred to a new 125ml shake flask. If the cells are aggregated, the cells need to be filtered by a cell screen before counting; on the day of transfection, 2ml centrifuge tubes were prepared and 0.8ml serum-free medium was added, respectively; one tube was filled with 30. Mu.g of plasmid and the other tube with 60. Mu.g PEI, gently tumbling the tube and mixing well several times; adding diluted PEI into diluted plasmids, slightly turning over a centrifuge tube, uniformly mixing for several times, and incubating for 15min at room temperature; slowly adding the mixture into 30ml of prepared cells, and shaking while adding to complete transfection; continuing to culture until the 6 th day for sample collection;
(4) Purification of the products of the expression plasmids Rituximab-pDAI, trastuzumab-pDAI, muromina b-CD3-pDAI and comparison of the expression levels with other double plasmid expression vectors
Centrifuging the transfected culture solution, filtering the supernatant with 0.22um membrane, dialyzing at 4deg.C to Na with concentration of 20mmol/mL and pH=7.0 containing 150mmol/mL NaCl 2 HPO 4 And (3) in the buffer solution, purifying by using a ProteinA column after the dialysis is finished, and obtaining the recombinant antibody protein.
Table 1 shows a comparison of the construction and expression processes of the recombinant expression vectors of the invention and other conventional expression vectors
Table 2 shows the comparison of the expression levels of the recombinant expression vectors of the present invention and other expression vectors.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. The expression vector of the recombinant antibody is characterized by specifically comprising an expression vector pDAI of an expression element of a mutant hCMV promoter, a chimeric Intron and a WPRE-BGH polyA terminator.
2. The method for preparing the recombinant antibody of interest expressed by the expression vector of the recombinant antibody according to claim 1, which comprises the following steps:
(1) Construction of expression vector pDAI
Sequence optimization design is carried out on sequence information to chemically synthesize a gene sequence (hCMV (M) -chimeric Intron) containing a mutant hCMV promoter and a chimeric Intron, and the gene sequence (WPRE-BGH polyA) of a WPRE and a BGH polyA terminator is chemically synthesized and connected to a pCDNA3.1 vector (enzyme cutting site Mlu I/Bbs I) framework which retains replicon and resistance genes through a homologous recombination method;
primer sequence:
F1：5‘-CGGGCCAGATATACGCGTTACATTGATTATTGACTAGTTATT-3’
F2：5‘-tccacaggtgtccagCGGATCCGAATTCTCGAaatcaacctct-3’
R1：5‘-agaggttgattTCGAGAATTCGGATCCGctggacacctgtgga-3’
R2：5‘-ATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTC-3’
first round PCR amplification system (total system 50 ul):
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 10s,30 cycles, holding at 16 ℃; amplifying a p1 fragment containing hCMV (M) -chimeric Intron; under the same conditions, replacing the upstream and downstream primers with F2 and R2, and amplifying a p2 fragment containing WPRE-BGH polyA;
second round PCR amplification system (overall 50 ul):
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10s, annealing at 65 ℃ for 5s, extension at 72 ℃ for 20s,30 cycles, holding at 16 ℃;
cloning the second round pcr product to a pCDNA3.1 vector with an enzyme cleavage site of Mlu I/Bbs I to construct an expression vector pDAI, and performing gel running and sequencing verification to obtain a successful vector construction;
(2) Construction of expression vectors Rituximab-pDAI, trastuzumab-pDAI, murominab-CD 3-pDAI
Chemically synthesizing a gene sequence containing a Kozak sequence, an antibody light chain of interest, P2A and an antibody heavy chain of interest, connecting the gene sequence to an expression vector pDAI (cloning site BamHI/EcoRI) by an enzyme digestion enzyme ligation method, and performing gel running and sequencing verification to prove that the vector is successfully constructed;
(3) Recombinant antibody expression plasmid Rituximab-pDAI, trastuzumab-pDAI, murominab-CD 3-pDAI extraction and CHO cell transfection, and other expression plasmid transfection steps are similar
Extracting: inoculating 10ml of bacteria according to an operation manual by using a Life plasmid DNA extraction kit, taking 2 mu L of the extracted plasmid, measuring the concentration by using a nucleic acid tester (Nanodrop One), taking 3 mu L of gel running electrophoresis detection and verification, and preserving the extracted plasmid at-20 ℃;
CHO cell transfection:
the day before transfection, cell count, 0.5X106/ml×30ml are transferred to a new 125ml shake flask, if the cells aggregate, the cell screen is needed to filter before counting; on the day of transfection, 2ml centrifuge tubes were prepared and 0.8ml serum-free medium was added, respectively; one tube was filled with 30. Mu.g of plasmid and the other tube with 60. Mu.g PEI, gently tumbling the tube and mixing well several times; adding diluted PEI into diluted plasmids, slightly turning over a centrifuge tube, uniformly mixing for several times, and incubating for 15min at room temperature; slowly adding the mixture into 30ml of prepared cells, and shaking while adding to complete transfection; continuing to culture until the 6 th day for sample collection;
(4) Purification of the products of the expression plasmids Rituximab-pDAI, trastuzumab-pDAI, muromina b-CD3-pDAI and comparison of the expression levels with other double plasmid expression vectors
Centrifuging the transfected culture solution, filtering the supernatant with 0.22um membrane, dialyzing at 4deg.C to Na with concentration of 20mmol/mL and pH=7.0 containing 150mmol/mL NaCl 2 HPO 4 And (3) in the buffer solution, purifying by using a ProteinA column after the dialysis is finished, and obtaining the recombinant antibody protein.
3. The method for preparing the recombinant antibody of interest expressed by the expression vector of the recombinant antibody according to claim 2, wherein the nucleotide sequence of hCMV (M) -chimeric Intron is:
ACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGGGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCCAGCggtttagtgaaccgtcagatcctgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccag。
4. the method for preparing the recombinant antibody of interest expressed by the expression vector of the recombinant antibody according to claim 3, wherein the nucleotide sequence of the WPRE-BGH polyA is:
AatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgTCTAGACGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG。
5. the method for preparing the recombinant antibody of interest expressed by the expression vector of the recombinant antibody according to claim 4, wherein the nucleotide sequence of Rituximab-pDAI is:
gccgccaccATGGTCCTCCAGACCCAGGTGTTCATCAGTCTGCTGCTGTGGATTAGCGGCGCCTACGGCCAGATCGTGCTGAGCCAGAGCCCCGCCATCCTGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATCCACTGGTTCCAGCAGAAGCCCGGCAGCAGCCCCAAGCCCTGGATCTACGCCACCAGCAACCTGGCCAGCGGCGTGCCCGTGCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCCGCGTGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGACCAGCAACCCCCCCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACCGCGGCGAGTGCGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGAAGCACCTGTGGTTCTTTCTGCTGCTCGTGGCTGCTCCCAGATGGGTCCTGAGCCAGGTGCAGCTGCAGCAGCCCGGCGCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACCCCCGGCCGCGGCCTGGAGTGGATCGGCGCCATCTACCCCGGCAACGGCGACACCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCAGCACCTACTACGGCGGCGACTGGTACTTCAACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCGCCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGCCGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGCTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG。
further, the nucleotide sequence of Trastuzumab-pDAI is:
gccgccaccATGGTCCTCCAGACCCAGGTGTTCATCAGTCTGCTGCTGTGGATTAGCGGCGCCTACGGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCCGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGCACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACCGCGGCGAGTGCGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGAAGCACCTGTGGTTCTTTCTGCTGCTCGTGGCTGCTCCCAGATGGGTCCTGAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGCATCTACCCCACCAACGGCTACACCCGCTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCAGCCGCTGGGGCGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGCGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGCTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG。
6. the method for preparing the recombinant antibody of interest expressed by the expression vector of the recombinant antibody according to claim 5, wherein the nucleotide sequence of the murominab-CD 3-pDAI is as follows:
gccgccaccATGGTCCTCCAGACCCAGGTGTTCATCAGTCTGCTGCTGTGGATTAGCGGCGCCTACGGCCAGATCGTTCTGACACAGAGCCCCGCTATCATGAGCGCTTCCCCAGGCGAGAAGGTGACCATGACCTGTAGCGCCTCTAGCAGCGTGTCTTACATGAACTGGTACCAGCAAAAGAGCGGCACAAGCCCTAAAAGATGGATCTACGACACCTCTAAACTGGCCAGCGGAGTGCCTGCCCACTTCCGGGGCAGCGGCAGCGGCACCTCTTATAGCCTGACCATCAGCGGTATGGAAGCTGAAGATGCCGCCACCTACTACTGCCAGCAATGGTCCAGCAACCCTTTTACCTTCGGCAGCGGCACAAAGCTGGAAATCAATAGAGCCGATACAGCCCCTACCGTGTCCATCTTCCCTCCCAGCAGCGAGCAGCTGACATCTGGAGGCGCCAGCGTGGTGTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGATCGACGGCTCTGAAAGACAGAACGGCGTGCTGAATAGCTGGACAGATCAGGACAGCAAGGACTCTACATACAGCATGTCCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACCTGCGAGGCCACACACAAGACCAGCACCTCCCCTATCGTGAAATCTTTTAACAGAAACGAGTGCGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGAAGCACCTGTGGTTCTTTCTGCTGCTCGTGGCTGCTCCCAGATGGGTCCTGAGCCAGGTGCAGCTGCAACAAAGCGGCGCCGAGCTGGCTAGACCTGGCGCTTCTGTGAAGATGAGCTGTAAAGCCAGCGGCTATACCTTTACCAGGTATACCATGCACTGGGTGAAGCAGCGGCCTGGCCAGGGCCTGGAATGGATCGGCTACATCAACCCCAGCCGGGGCTACACAAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACTGATAAGAGCTCTTCTACAGCCTACATGCAGCTGTCTTCTCTGACAAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTATTGTCTGGATTACTGGGGCCAGGGCACCACCCTGACCGTGTCCAGCgctagcGCAAAGACGACCGCCCCCAGCGTGTACCCTCTGGCCCCTGTGTGCGGAGGCACCACAGGCAGCTCCGTGACACTGGGCTGTCTGGTGAAAGGCTACTTCCCTGAACCCGTGACCCTGACCTGGAACAGCGGTAGCCTGTCCTCCGGAGTTCATACATTCCCCGCCGTGCTGCAGAGCGATCTGTACACACTGAGCAGCTCTGTGACCGTGACCAGCAGCACATGGCCTAGCCAGAGCATTACATGCAACGTGGCCCACCCCGCCAGCAGCACCAAGGTGGACAAAAAGATCGAGCCCAGACCAAAGAGCTGCGACAAAACACACACCTGCCCTCCTTGCCCCGCCCCAGAACTGCTGGGGGGCCCTTCTGTGTTCCTGTTTCCACCTAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAGGTGACCTGCGTGGTGGTCGACGTGAGCCATGAGGACCCTGAGGTGAAATTCAACTGGTACGTGGACGGCGTGGAAGTCCACAACGCCAAGACAAAGCCCCGGGAAGAGCAGTACAATAGCACCTACAGAGTGGTGTCTGTCCTGACCGTTCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAGACAATCTCCAAAGCCAAGGGCCAGCCTAGAGAGCCCCAGGTGTACACCCTGCCTCCTTCCCGCGACGAGCTGACAAAGAACCAGGTGTCCCTCACCTGTCTGGTGAAAGGCTTCTACCCCAGCGACATCGCCGTGGAATGGGAGAGCAACGGACAACCTGAAAACAACTACAAGACCACCCCTCCAGTGCTGGACTCCGATGGCTCCTTCTTCCTCTACAGCAAGCTGACCGTGGATAAGTCTCGGTGGCAGCAGGGAAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGCCTGTCACCTGGAAAGTAAGAATTC。
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