阅读说明：本技术 一种提高IgG1抗体产量的方法 (Method for improving IgG1 antibody yield ) 是由 赵妍淑 张金华 宋浩 于 2019-08-19 设计创作，主要内容包括：本发明公开了一种提高IgG1抗体产量的方法,包括如下步骤：(1)基因的体外合成和扩增：得到优化后的lgG1的轻链和重链的核苷酸序列,连接成一条核苷酸序列；(2)将核苷酸序列连接到p2A4载体中得到p2A4-HL；(3)sRNA的设计和组装：(4)分别将连接到sRNA接收体的sRNA序列连接到p3C5载体；(5)分别与p2A4-HL一起导入大肠杆菌中,得到重组菌1、重组菌2、重组菌3、重组菌4和重组菌5；(6)将重组菌分别发酵,得到高产量IgG1抗体。本发明的方法能提高单克隆抗体在大肠杆菌中的表达量,针对性的抑制三羧酸循环代谢路径的相关酶,会使用来合成冗余酶的氨基酸更多的合成IgG1抗体,从而增加IgG1的产量。(The invention discloses a method for improving the yield of an IgG1 antibody, which comprises the following steps: (1) in vitro synthesis and amplification of genes: obtaining optimized nucleotide sequences of the light chain and the heavy chain of lgG1, and connecting the nucleotide sequences into a nucleotide sequence; (2) connecting the nucleotide sequence into a p2A4 vector to obtain p2A 4-HL; (3) design and assembly of sRNA: (4) ligating the sRNA sequences linked to the sRNA acceptor to p3C5 vectors, respectively; (5) respectively introducing the recombinant strain and p2A4-HL into escherichia coli together to obtain a recombinant strain 1, a recombinant strain 2, a recombinant strain 3, a recombinant strain 4 and a recombinant strain 5; (6) and (3) fermenting the recombinant bacteria respectively to obtain the high-yield IgG1 antibody. The method can improve the expression amount of the monoclonal antibody in the escherichia coli, and the target enzyme for inhibiting the metabolic pathway of the tricarboxylic acid cycle can synthesize more IgG1 antibodies by using more amino acids for synthesizing redundant enzymes, thereby increasing the yield of IgG 1.)

1. A method for improving the production of IgG1 antibody, comprising the steps of:

(1) in vitro synthesis and amplification of genes:

the light chain of lgG1 and the heavy chain of lgG1 derived from cetuximab were obtained from the NCBI database, the amino acid sequences of the light and heavy chains of lgG1 were respectively at the JCAT website by entering: obtaining nucleotide sequences of an optimized lgG1 light chain and an optimized lgG1 heavy chain under the condition of avoiding restriction sites of XbaI, NdeI, SpeI and HindIII, and synthesizing and amplifying in vitro; connecting the optimized light chain of lgG1 and the optimized heavy chain into a nucleotide sequence; the amino acid sequence of the light chain of lgG1 is shown in SEQ ID NO. 1; the amino acid sequence of the heavy chain of lgG1 is shown in SEQ ID NO. 3; the nucleotide sequence of the optimized lgG1 light chain is shown in SEQ ID NO. 2; the nucleotide sequence of the optimized lgG1 heavy chain is shown as SEQ ID NO. 4;

(2) connecting the nucleotide sequence obtained in the step (1) into a p2A4 vector to obtain p2A 4-HL; the nucleotide sequence of the p2A4 vector is shown in SEQ ID NO. 31;

(3) design and assembly of sRNA: the 5 relevant gene sequences in the E.coli tricarboxylic acid pathway were selected and listed on the JCAT site by entering: avoiding restriction sites of XbaI, NdeI, SpeI and HindIII as conditions to obtain optimized nucleotide sequences of 5 related genes of gltA, sdhA, sucB, sucD and fumC; the nucleotide sequence of the optimized gltA is shown as SEQ ID NO. 5; the nucleotide sequence of the optimized sdhA is shown as SEQ ID NO. 6; the nucleotide sequence of the optimized sucB is shown as SEQ ID NO. 7; the nucleotide sequence of the optimized sucD is shown as SEQ ID NO. 8; the nucleotide sequence of the optimized fumC is shown in SEQ ID NO. 9; respectively selecting 20-30nt oligonucleotides as templates from the ATG of the optimized 5 related gene nucleotide sequences, designing a forward primer and a reverse primer, adding a viscous tail end TTGC at the 5 'end of the forward primer, adding a viscous tail end GAAA at the 5' end of the reverse primer, measuring the binding free energy through a free energy detection website, enabling the binding free energy to be between-30 and-40 kcal/mol, and obtaining 5 nucleotide sequences, namely sRNA-1, sRNA-2, sRNA-3, sRNA-4 and sRNA-5, which are reversely complementary with the 20-30nt oligonucleotides through PCR; using the golden gate method to join 5 sRNA sequences to a sRNA acceptor with a nucleotide sequence shown in SEQ ID No.20 to give 5 nucleotide sequences;

(4) respectively connecting the sRNA sequences connected to the sRNA acceptor to a p3C5 vector to obtain p3C5-gltA, p3C5-sdhA, p3C5-sucB, p3C5-sucD and p3C 5-fumC; the nucleotide sequence of the p3C5 vector is shown as SEQ ID NO. 32;

(5) respectively introducing p3C5-gltA, p3C5-sdhA, p3C5-sucB, p3C5-sucD and p3C5-fumC together with p2A4-HL into escherichia coli to sequentially obtain a recombinant bacterium 1, a recombinant bacterium 2, a recombinant bacterium 3, a recombinant bacterium 4 and a recombinant bacterium 5;

(6) and respectively fermenting the recombinant bacterium 1, the recombinant bacterium 2, the recombinant bacterium 3, the recombinant bacterium 4 and the recombinant bacterium 5 to obtain the high-yield IgG1 antibody.

Technical Field

The invention relates to the field of biology, and particularly relates to a method for improving the yield of an IgG1 antibody.

Background

At present, methods for mass production of monoclonal antibodies are mainly divided into three categories, one is an in vivo method, i.e., hybridoma cells are injected into the abdominal cavity of a genotype-compatible animal to obtain monoclonal antibodies from ascites; the second is an in vitro method, namely, a monoclonal antibody is produced by culturing a large amount of hybridoma cells in vitro; thirdly, the gene engineering antibody technology, namely, the antibody gene in the monoclonal antibody hybridoma is cloned through the tombardin operation technology or the antibody gene is directly obtained through the antibody library technology and then is expressed in other expression vectors in large quantity. However, hybridoma cells are not suitable for all laboratories, and mammalian cells have poor tolerance and high production cost. The Escherichia coli expression system has the characteristics of large scale, high speed, low cost and the like, and becomes a convenient and rapid production system. However, at present, the yield is optimized only from the fermentation process and the monoclonal antibody itself, and the influence of the tricarboxylic acid cycle pathway of the escherichia coli (as shown in fig. 1) on the yield of the monoclonal antibody is not studied.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for improving the yield of IgG1 antibody.

The technical scheme of the invention is summarized as follows:

a method of increasing the production of IgG1 antibody, comprising the steps of:

(1) in vitro synthesis and amplification of genes:

the light chain of lgG1 and the heavy chain of lgG1 derived from cetuximab were obtained from the NCBI database, the amino acid sequences of the light and heavy chains of lgG1 were respectively at the JCAT website by entering: obtaining nucleotide sequences of an optimized lgG1 light chain and an optimized lgG1 heavy chain under the condition of avoiding restriction sites of XbaI, NdeI, SpeI and HindIII, and synthesizing and amplifying in vitro; connecting the optimized light chain of lgG1 and the optimized heavy chain into a nucleotide sequence; the amino acid sequence of the light chain of lgG1 is shown in SEQ ID NO. 1; the amino acid sequence of the heavy chain of lgG1 is shown in SEQ ID NO. 3; the nucleotide sequence of the optimized lgG1 light chain is shown in SEQ ID NO. 2; the nucleotide sequence of the heavy chain of the optimized lgG1 is shown in SEQ ID NO. 4;

(2) connecting the nucleotide sequence obtained in the step (1) into a p2A4 vector to obtain p2A 4-HL; the nucleotide sequence of the p2A4 vector is shown in SEQ ID NO. 31;

(3) design and assembly of sRNA: the 5 relevant gene sequences in the E.coli tricarboxylic acid pathway were selected and listed on the JCAT site by entering: avoiding restriction sites of XbaI, NdeI, SpeI and HindIII as conditions to obtain optimized nucleotide sequences of 5 related genes of gltA, sdhA, sucB, sucD and fumC; the nucleotide sequence of the optimized gltA is shown as SEQ ID NO. 5; the nucleotide sequence of the optimized sdhA is shown as SEQ ID NO. 6; the nucleotide sequence of the optimized sucB is shown as SEQ ID NO. 7; the nucleotide sequence of the optimized sucD is shown as SEQ ID NO. 8; the nucleotide sequence of the optimized fumC is shown in SEQ ID NO. 9; respectively selecting 20-30nt oligonucleotides as templates from the ATG of the optimized 5 related genes, designing a forward primer and a reverse primer, adding a viscous tail end TTGC to the 5 'end of the forward primer, adding a viscous tail end GAAA to the 5' end of the reverse primer, measuring the binding free energy through a free energy detection website, enabling the binding free energy to be between-30 and-40 kcal/mol, and obtaining 5 nucleotide sequences which are reversely complementary with the 20-30nt oligonucleotides through PCR, wherein the 5 nucleotide sequences are respectively sRNA-1, sRNA-2, sRNA-3, sRNA-4 and sRNA-5; using the golden gate method to join 5 sRNA sequences to a sRNA acceptor with a nucleotide sequence shown in SEQ ID No.20 to give 5 nucleotide sequences;

(4) respectively connecting the sRNA sequences connected to the sRNA acceptor to a p3C5 vector to obtain p3C5-gltA, p3C5-sdhA, p3C5-sucB, p3C5-sucD and p3C 5-fumC; the nucleotide sequence of the p3C5 vector is shown as SEQ ID NO. 32;

(5) respectively introducing p3C5-gltA, p3C5-sdhA, p3C5-sucB, p3C5-sucD and p3C5-fumC together with p2A4-HL into escherichia coli to sequentially obtain a recombinant bacterium 1, a recombinant bacterium 2, a recombinant bacterium 3, a recombinant bacterium 4 and a recombinant bacterium 5;

(6) and respectively fermenting the recombinant bacterium 1, the recombinant bacterium 2, the recombinant bacterium 3, the recombinant bacterium 4 and the recombinant bacterium 5 to obtain the high-yield IgG1 antibody.

The invention has the advantages that:

the method can improve the expression amount of the monoclonal antibody in the escherichia coli, and the target enzyme for inhibiting the metabolic pathway of the tricarboxylic acid cycle can synthesize more IgG1 antibodies by using more amino acids for synthesizing redundant enzymes, thereby increasing the yield of IgG 1.

Drawings

FIG. 1 shows the tricarboxylic acid cycle pathway of E.coli itself.

FIG. 2 is a graph showing the results of the concentrations of IgG1 produced by fermentation of 5 different recombinant bacteria.

Detailed Description

The present invention will be further described with reference to the following examples.