Recombinant host cell and method for producing nano antibody by using same

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant host cell and a method for producing a nano antibody by using the recombinant host cell. The recombinant host cell disclosed by the invention contains coding genes of the nano antibody and the auxiliary protein; the auxiliary proteins are i) a transcriptional regulation protein Hac1 and a heavy chain binding protein Bip; and / or ii) protein disulfide bond isomerase Pdi and endoplasmic reticulum redox protein Ero1; coding genes of the nano antibody and the auxiliary protein exist in a host cell in the form of an expression vector or are integrated in a genome of the host cell. The invention provides a novel nano antibody expression system through a co-expression auxiliary protein strategy, and the expression system has the advantages of simplicity, economy, high yield, high purity and the like, and is suitable for industrial production and research application. The method can be used for rapidly and efficiently producing the nano antibody, and provides important technical support for antibody drug development and research in the field of biomedicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant host cell and a method for producing nano antibodies using the same. Background Art

[0002] Nanobodies are a type of heavy chain antibody variable region that is naturally missing light chains and is found by scientists in 1989. The molecular weight is only 15 ku, which is 1 / 10 of the molecular weight of conventional antibodies. The length of its protein crystal structure is 4 nm and the diameter is 2.5 nm. It is the antibody with the smallest molecular weight known so far, so it is called nanobodies. Nanobodies have many advantages, including small size, high affinity and specificity, stability, easy expression and easy engineering. Its small size enables it to better penetrate into tissues and cells to achieve better targeting effects; its high affinity and specificity enable it to perform well in targeted therapy and diagnosis; its stability enables it to maintain activity in complex biological environments; its easy expression means that it can be efficiently expressed using a variety of expression systems, including bacteria, yeast, insect cells, mammalian cells, etc.; and its easy engineering allows it to be further modified to meet the needs of different applications. Nanobodies have shown a wide range of application potential in the fields of tumor therapy, biosensors, immunohistochemistry, molecular imaging, etc. As a new type of antibody molecule, nanobodies provide new possibilities for precision medicine and personalized treatment, and provide new tools and methods for biomedical research and clinical diagnosis.

[0003] CN 115819599 A discloses a method for secretory expression of nano antibody Cablivi using recombinant Escherichia coli, comprising the following steps: (1) constructing a recombinant Escherichia coli containing a target gene of nano antibody Cablivi; (2) culturing and inducing secretory expression of the recombinant Escherichia coli; (3) extracting nano antibody Cablivi from the periplasmic space. The final yield of nano antibody Cablivi extracted by this method can reach 17.6 mg / L.

[0004] CN117720649A discloses that a heavy chain antibody variable region sequence against H9 subtype avian influenza virus was screened from an established immune alpaca VHH phage library, the sequence was directionally cloned into a eukaryotic expression plasmid pPIC9K to construct a recombinant eukaryotic expression plasmid, and the plasmid was transformed into a Pichia yeast expression system to screen the expression engineering strain, and the recombinant nano antibody was obtained after methanol induction, supernatant collection, and affinity chromatography purification. The yield of the recombinant nano antibody reaches 3.5 mg / L.

[0005] CN117417441A discloses that a heavy chain antibody variable region sequence against Newcastle disease virus was screened from an established immune alpaca VHH phage library, the sequence was directionally cloned into a eukaryotic expression plasmid pPIC9K to construct a recombinant eukaryotic expression plasmid, and the plasmid was transformed into a Pichia yeast expression system to screen the expression engineering strain, and the recombinant nano antibody was obtained after methanol induction, supernatant collection, and affinity chromatography purification. The yield of the recombinant nano antibody reaches 15 mg / L.

[0006] The existing methods for preparing nanobodies through genetic transformation have low overall yields. The methanol-induced expression system has many problems such as the danger and high cost of storing and transporting large amounts of methanol and a long fermentation cycle. Summary of the invention

[0007] In view of the above-mentioned problems existing in the prior art, the present invention provides a recombinant host cell and a method for producing nanobodies using the same, which can significantly improve the expression efficiency and stability of nanobodies and simplify downstream separation and purification, while shortening the fermentation cycle and reducing production costs.

[0008] In the first aspect, the present invention provides a recombinant host cell, which contains genes encoding nano antibodies and auxiliary proteins; the auxiliary proteins are: i) transcriptional regulatory protein Hac1 and heavy chain binding protein Bip; and / or, ii) protein disulfide isomerase Pdi and endoplasmic reticulum redox protein Ero1; the genes encoding nano antibodies and auxiliary proteins are present in the host cell in the form of expression vectors, or are integrated into the genome of the host cell.

[0009] In some embodiments, the recombinant host cell contains a first expression vector, a second expression vector and / or a third expression vector; the first expression vector contains a first expression cassette for expressing a nanobody; the second expression vector contains a second expression cassette for expressing the transcriptional regulatory protein Hac1 and a third expression cassette for expressing the heavy chain binding protein Bip; the third expression vector contains a fourth expression cassette for expressing the protein disulfide isomerase Pdi and a fifth expression cassette for expressing the endoplasmic reticulum redox protein Ero1.

[0010] In some embodiments, the promoter of the first expression cassette, the second expression cassette, the third expression cassette, the fourth expression cassette, or the fifth expression cassette is P Gap Promoter.

[0011] In some embodiments, the gene encoding the Nanobody has a nucleotide sequence as shown in SEQ ID NO.1.

[0012] In some embodiments, the gene encoding the transcriptional regulatory protein Hac1 has a nucleotide sequence as shown in SEQ ID NO.2.

[0013] In some embodiments, the gene encoding the heavy chain binding protein Bip has a nucleotide sequence as shown in SEQ ID NO.3.

[0014] In some embodiments, the gene encoding protein disulfide isomerase Pdi has a nucleotide sequence as shown in SEQ ID NO.4.

[0015] In some embodiments, the gene encoding the endoplasmic reticulum redox protein Ero1 has a nucleotide sequence as shown in SEQ ID NO.5.

[0016] In some embodiments, the host cell is a bacterial cell, a yeast cell, or a mammalian cell, preferably a Pichia pastoris cell.

[0017] In some embodiments, the base vector of the expression vector is a vector suitable for expression in Pichia pastoris cells.

[0018] In a second aspect, the present invention provides a method for producing a nanobody, comprising: constructing the aforementioned recombinant host cell; culturing the recombinant host cell; and isolating and obtaining the nanobody from the culture product.

[0019] Pichia pastoris Pichia pastoris ) is a commonly used fungal expression system that is widely used in the fields of biotechnology and biomedicine. It has many advantages that make it an ideal choice for protein expression and industrial production. First, Pichia pastoris has efficient protein expression capabilities. Its genome contains a wealth of transcriptional regulatory elements and secretion pathway-related genes, which can achieve high levels of target protein expression and secretion. In addition, Pichia pastoris has a fast cell growth rate and relatively simple culture conditions, which is convenient for large-scale production. Secondly, Pichia pastoris has good soluble protein expression capabilities. Due to the characteristics of its secretion pathway, the target protein is usually expressed in a soluble form in the culture supernatant, which reduces protein aggregation and the formation of inclusion bodies, and is conducive to subsequent purification and functional studies. In addition, Pichia pastoris has strong genetic engineering performance. Through genetic engineering methods, Pichia pastoris can be subjected to gene knockout, gene overexpression, protein labeling and other operations to meet the needs of different research and applications. In addition, the genome sequence of Pichia pastoris has been fully determined, which provides convenience for further genetic engineering and metabolic engineering. Finally, Pichia pastoris performs well in terms of biosafety. Compared with other commonly used fungal expression systems, such as Saccharomyces cerevisiae ( Saccharomyces cerevisiae), Pichia pastoris has little impact on human health and the environment, and has low pathogenicity and infectious risks. Therefore, as a reliable and efficient fungal expression system, Pichia pastoris plays an important role in protein expression and industrial production. Its advantages include efficient protein expression ability, good soluble protein expression ability, strong genetic engineering performance and good biosafety. This makes Pichia pastoris an important tool and platform widely used in biotechnology and biomedicine.

[0020] In recent years, Pichia pastoris has been recognized as an important platform for the production of various heterologous proteins. The strong methanol-induced promoter AOX1, with the help of the α-pre-pro signal sequence, can lead to high expression levels of extracellular proteins. GAP It is a strong promoter of constitutive expression, which can efficiently express foreign proteins in culture medium containing various carbon sources such as glycerol and glucose. GAP ) expression level and P AOX1 The expression levels of GAP The expression system is more suitable for large-scale industrial production of foreign proteins because it can avoid the danger and high cost of storing and transporting large amounts of methanol, and does not require carbon source conversion during the fermentation process, which greatly shortens the fermentation cycle. However, the promoter replacement strategy is not always effective because P. pastoris Protein secretion in the mitochondria involves a number of programs mediated by several cellular proteins, including folding assisted by endoplasmic reticulum (ER) chaperones, degradation by ubiquitination, and an efficient vesicular transport system. Efficient protein expression requires the cooperation of various intracellular pathways.

[0021] When the gene P. pastorisWhen overexpressed in the endoplasmic reticulum, a large number of newly formed peptide chains may aggregate and remain in the endoplasmic reticulum cavity due to the inability to fold correctly in time, thus causing huge endoplasmic reticulum pressure. At this time, the misfolded protein will competitively bind to the heavy chain binding protein Bip, causing the endoplasmic reticulum pressure sensing factor Ire1p (transmembrane kinase / ribonuclease) originally bound to Bip to be released from the Bip / Ire1p complex and aggregated in the endoplasmic reticulum to form oligomers, causing Ire1p to autophosphorylate and exhibit endoribonuclease activity. Subsequently, Ire1p completes the splicing of Hac1 gene mRNA with the assistance of tRNA ligase and makes it express active Hac1 protein, resulting in an unfolded protein response (UPR). As a UPR transcription activator, Hac1 (transcriptional regulatory protein) is transported to the nucleus and relies on UPR recognition elements (UPRE) to recognize and regulate the expression of many partner genes involved in protein folding and transport, endoplasmic reticulum quality control (ER quality control) and endoplasmic reticulum-associated protein degradation (ERAD), thereby alleviating the endoplasmic reticulum pressure caused by a large number of newly formed peptide chains that cannot be folded correctly and timely and are aggregated and retained in the endoplasmic reticulum cavity, which is crucial for the formation of the correct protein conformation. Bip (heavy chain binding protein) can not only bind to unfolded proteins to promote the cell's sensing of ER pressure, but also help the correct folding of newly formed peptide chains; Pdi (protein disulfide isomerase) can catalyze the formation and isomerization of protein disulfide bonds, and also plays an important role in the folding process of newly formed proteins; Ero1 (endoplasmic reticulum redox protein) is responsible for the redox state of Pdi so that it can continue to participate in the folding of auxiliary proteins (because Pdi will be reduced after catalyzing the formation of disulfide bonds in proteins). Therefore, a strategy of co-expressing auxiliary proteins such as Hac1 (transcriptional regulatory protein), Bip (heavy chain binding protein), Pdi (protein disulfide isomerase), and Ero1 (endoplasmic reticulum redox protein) was proposed to increase protein folding and processing capabilities to achieve efficient secretion involving overexpression of nanoantibodies.

[0022] The beneficial effects of the present invention are: The recombinant host cell provided by the present invention co-expresses nano antibodies with auxiliary proteins selected from the group consisting of transcriptional regulatory protein Hac1, heavy chain binding protein Bip, protein disulfide isomerase Pdi and endoplasmic reticulum redox protein Ero1 in host cells for the first time, thereby providing a new nano antibody efficient expression system.

[0023] The method for producing nanobodies provided by the present invention utilizes P GAPThe constitutive promoter avoids the induction of methanol, and combined with the strategy of co-expressing auxiliary proteins, it realizes the simple, economical, high-yield (shake flask protein expression reaches 0.18 g / L), high-purity (purity reaches 60%) nanoantibody production. This method has important application value and can be used for antibody drug development and biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Prepare flow charts for host 1, host 2, host 3, and host 4; Figure 2 is the pNb plasmid map; Figure 3 This is the pAP1 plasmid map; Figure 4 This is the pAP2 plasmid map; Figure 5 This is the growth curve of host 1 strains with different copy numbers; Figure 6 SDS-PAGE results of supernatant samples of host 1 fermentation broth with different copy numbers-3d; Figure 7 SDS-PAGE results of supernatant samples of host 1 fermentation broth with different copy numbers -4d; Figure 8 These are the growth curves of the strains of the empty host, host 1, host 2, host 3, and host 4; Fig. 9 SDS-PAGE results of the fermentation supernatant samples of the empty host, host 1, host 2, host 3, and host 4 -72h; Fig.10 SDS-PAGE results of fermentation supernatant samples of empty host, host 1, host 2, host 3, and host 4 - 96h; Fig.11 Comparison of protein expression levels of empty host, host 1, host 2, host 3, and host 4 strains. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1 Design and construction of plasmids for co-expressing auxiliary proteins and plasmids for expressing nanobodies The plasmid pGAPZαA-His-Cablivi (abbreviated as pNb) for expressing nanobodies was constructed. The plasmid backbone was pGAPZαA and the promoter was P Gap The signal peptide is α-factor, the expressed protein is the bivalent nanoantibody Cablivi, and a His tag is added to the N-terminus of the target protein, and the screening marker bleomycin is used; the recombinant plasmid was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. to construct, and the pNb plasmid map can be found at Figure 2 The successfully synthesized plasmid was transformed into the E. coli DH5α strain according to conventional methods. The specific sequence of the nanobody Cablivi is as follows (SEQ ID NO.1): GAGGTTCAATTGGTTGAATCCGGGTGGTGGTTTGGTTCAACCAGGTGGTTCTTTGAGATTGTCCTGTGCTGCTTCCGGTAGAACCTTCTCTTACAATCCAATGGGTTGGTTCAGACAGGCTCCAGGTAAGGGTAGAGAATTGGTTGCTGCTATCTCCAGAACTGGTGGTTCCACTTACTACCCAGACTCTGTTGAG GGTAGATTCACCATCTCCAGAGACAACGCCAAGAGAATGGTCTACTTGCAGATGAACTCCTTGAGAGCTGAGGACACTGCCGTTTACTATTGTGCTGCTGCTGGTGTTAGAGCCGAGGATGGTAGAGTTAGAACTTTGCCATCCGAGTACACTTTCTGGGGTCAAGGTACTCAGGTTACTGTTTCTTCTGCTGCT GCCGAAGTTCAGTTGGTAGAAAGTGGTGGTGGACTTGTTCAGCCTGGTGGATCCTTGAGACTTTTCTTGTGCAGCATCCGGTCGTACTTTCAGCTATAACCCTATGGGATGGTTTAGACAAGCCCCTGGTAAAGGTCGTGAATTGGTCGCAGCAATTTCAAGAACCGGCGGATCTACTTACTATCCTGATTCCGTC GAAGGCAGGTTCACTATTTCCAGGGATAACGCTAAGCGTATGGTGTACCTTCAAATGAACTCTTTGCGTGCCGAGGATACCGCTGTTTATTACTGTGCAGCCGCAGGCGTTAGAGCTGAAGATGGAAGGGTAAGAACACTGCCCTCTGAGTATACTTTTTGGGGACAGGGAACCCAAGTTACCGTTTCCTCTTAA Carrying two independent expression cassettes to express Hac1 and Bip auxiliary proteins respectively, the plasmid backbone is pPIC3.5, and the promoter is replaced by P Gap , the screening marker is histidine, set as plasmid pAP1, which will help the folding and processing of exogenous target proteins and improve the expression efficiency of exogenous proteins; the recombinant plasmid was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. to construct, and the pAP1 plasmid map can be found at Figure 3 The successfully synthesized plasmid was transformed into the E. coli DH5α strain according to conventional methods. The specific sequence of Hac1 is as follows (SEQ ID NO.2): ATGCCCGTAGATTCTTCTCATAAGACAGCTAGCCCACTTCCACCTCGTAAAAGAGCAAAGACGGAAGAAGAAAAGGAGCAGCGTCGAGTGGAACGTATCCTACGTAATAGGAGAGCGGCTCATGCTTCCAGAGAGAAGAAACGAAGACACGTTGAATTTCTGGAGAACCACGTCGTCGA T CTGGAATCTGCACTTCAAGAATCAGCCAAAGCCACTAACAAGTTGAAAGAAATACAAGATATCATTGTTTCAAGGTTGAAAGCCTTAGGTGGTACCGTCTCAGATTTGGATTTAACAGTTCCGGAAGTCGATTTTCCCAAATCTTCTGATTTGGAACCCATGTCTGATCTCTCAACCTCTTCGAAATCGGAGAAAGCATCTACATCCACTCGCAGATCTTTGACTGAGGATCTGGACGAAGATGACGTCGCTGAATATGACGACGAAGAAGAGGACGAAGAGTTACCCAGGAAAATGAAAGTCTTAAACGACAAAAACAAGAGCACATCTATCAAGCAGGAGAAGTTGAATGAACTTCCATCTCCTTTGTCATCCGATTTTTCAGACGTAGATGAAGAAAGGTCAACTCTCACACATTTAAAGTTGCAACAGCAACAACAACAACCAGTAGACAATTATGTTTCTACTCCTTTGAGTCTTCCGGAGGATTCAGTTGATTTTATTAACCCAGGTAACTTAAAAATAGAGTCCGATGAGAACTTCTTGTTGAGTTCAAATACTTTACAAATAAAACACGAAAAGGACACCGAGTACATTACTACAGCTCCATCAGGTTCCATCAATGATTTTTTTAATCCTTATGACATTAGCGAGTCGAATCGGTTGCATCATCCAGCAGCACCATTTACCGCTAATGCATCTGATTTAAATGACTTTGTATTCTTCCAGGAATAG * The underlined part is a synonymous mutation to remove the Sal I site The specific sequence of Bip is as follows (SEQ ID NO.3): ATGCTGTCGTTAAAACCATCTTGGCTGACTTTGGCGGCATTAATGTATGCCATGCTATTGGTCGTAGTGCCATTTGCTAAACCTGTTAGAGCTGACGATGTCGAATCTTATGGAACAGTGATTGGTATCGATTTGGGTACCACGTACTCTTGTGTCGGTGTGATGAAGTCGGGTCGTGTAGAAATTCTTGCTAATGACCAAGGTAACAGAATCACTCCTTCCTACGTTAGTTTCACTGAAGACGAGAGACTGGTTGGTGATGCTGCTAAGAACTTAGCTGCTTCTAACCCAAAAAACACCATCTTTGATATTAAGAGATTGATCGGTATGAAGTATGATGCCCCAGAGGTCCAAAGAGACTTGAAGCGTCTTCCTTACACTGTCAAGAGCAAGAACGGCCAACCTGTCGTTTCTGTCGAGTACAAGGGTGAGGAGAAGTCTTTCACTCCTGAGGAGATTTCCGCCATGGTCTTGGGTAAGATGAAGTTGATCGCTGAGGACTACTTAGGAAAGAAAGTCACTCATGCTGTCGTTACCGTTCCAGCCTACTTCAACGACGCTCAACGTCAAGCCACTAAGGATGCCGGTCTCATCGCCGGTTTGACTGTTCTGAGAATTGTGAACGAGCCTACCGCCGCTGCCCTTGCTTACGGTTTGGACAAGACTGGTGAGGAAAGACAGATCATCGTCTACGACTTGGGTGGAGGAACCTTCGATGTTTCTCTGCTTTCTATTGAGGGTGGTGCTTTCGAGGTTCTTGCTACCGCCGGTGACACCCACTTGGGTGGTGAGGACTTTGACTACAGAGTTGTTCGCCACTTCGTTAAGATTTTCAAGAAGAAGCATAACATTGACATCAGCAACAATGATAAGGCTTTAGGTAAGCTGAAGAGAGAGGTCGAAAAGGCCAAGCGTACTTTGTCTTCCCAGATGACTACCAGAATTGAGATTGACTCTTTCGTCGAT GGTATCGACTTCTCTGAGCAACTGTCTAGAGCTAAGTTTGAGGAGATCAACATTGAATTATTCAAGAAGACACTGAAACCAGTTGAACAAGTCCTCAAAGACGCTGGTGTCAAGAAATCTGAAATTGATGACATTGTCTTGGTTGGTGGTTCTACCAGAAT CCCAAAGGTTCAACAATTATTGGAGGATTACTTTGACGGAAAGAAGGCTTCTAAGGGAATTAACCCAGATGAAGCTGTCGCATACGGTGCTGCTGTTCAGGCTGGTGTTTTGTCTGGTGAGGAAGGTGTCGATGACATCGTCTTGCTTGATGTGAACCCCCTAACTCTGGGTATCGAGACTACTGGTGGCGTTATGACTACCTTAATCAACAGAAACACTGCTATCCCAACTAAGAAATCTCAAATTTTCTCCACTGCTGCTGACAACCAGCCAACTGTGTTGATTCAAGTTTATGAGGGTGAGAGAGCCTTGGCTAAGGACAACAACTTGCTTGGTAAATTCGAGCTGACTGGTATTCCACCAGCTCCAAGAGGTACTCCTCAAGTTGAGGTTACTTTTGTTTTAGACGCTAACGGAATTTTGAAGGTCTCTGCCACCGATAAGGGAACTGGAAAATCCGAGTCCATCACCATCAACAATGATCGTGGTAGATTGTCCAAGGAGGAGGTTGACCGTATGGTTGAAGAGGCCGAGAAGTACGCCGCTGAGGATGCTGCACTAAGAGAAAAGATTGAGGCTAGAAACGCTCTGGAGAACTACGCTCATTCCCTTAGGAACCAAGTTACTGATGACTCTGAAACCGGGCTTGGTTCTAAATTGGACGAGGACGACAAAGAGACATTGACAGATGCCATCAAAGATACCCTAGAGTTCTTGGAAGACAACTTCGACACCGCAACCAAGGAAGAATTAGACGAACAAAGAGAAAAGCTTTCCAAGATTGCTTACCCAATCACTTCTAAGCTATACGGTGCTCCAGAGGGTGGTACTCCACCTGGTGGTCAAGGTTTTGACGATGATGATGGAGACTTTGACTACGACTATGACTATGATCATGATGAGTTGTAG * The underlined part is a synonymous mutation to remove the Sal I and EcoR I sites Carrying two independent expression cassettes to express Pdi and Ero1 auxiliary proteins respectively, the plasmid backbone is pPIC3.5K, and the promoter is replaced by P Gap , the selection marker is geneticin, set as plasmid pAP2, which will help catalyze the formation of protein disulfide bonds and improve the expression efficiency of foreign proteins; the recombinant plasmid was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. to construct, and the pAP2 plasmid map can be found in Figure 4 The successfully synthesized plasmid was transformed into the E. coli DH5α strain according to conventional methods. The specific sequence of Pdi is as follows (SEQ ID NO.4): ATGCAATTCAACTGGAATATTAAAACTGTGGCAAGTATTTTGTCCGCTCTCACACTAGCACAAGCAAGTGATCAGGAGGCTATTGCTCCAGAGGACTCTCATGTCGTCAAATTGACTGAAGCCACTTTTGAGTCTTTCATCACCAGTAATCCTCACGTTTTGGCAGAGTTTTTTGCCCCTTGGTGTGGTCA CTGTAAGAAGTTGGGCCCTGAACTTGTTTCTGCTGCCGAGATCTTAAAGGACAATGAGCAGGTTAAGATTGCTCAAATTGATTGTACGGAGGAGAAGGAATTATGTCAAGGCTACGAAATTAAAGGGTATCCTACTTTGAAGGTGTTCCATGGTGAGGTTGAGGTCCCAAGTGACTATCAAGGTCAAAGAC AGAGCCAAAGCATTGTCAGCTATATGCTAAAGCAGAGTTTACCCCCTGTCAGTGAAATCAATGCAACCAAAGATTTAGACGACACAATCGCCGAGGCAAAAGAGCCCGTGATTGTGCAAGTACTACCGGAAGATGCATCCAACTTGGAATCTAACACCACATTTTACGGAGTTGCCGGTACTCTCAGAGAG AAATTCACTTTTGTCTCCACTAAGTCTACTGATTATGCCAAAAAATACACTAGCGACTCGACTCCTGCCTATTTGCTTGTCAGACCTGGCGAGGAACCTAGTGTTTACTCTGGTGAGGAGTTAGATGAGACTCATTTGGTGCACTGGATTGATATTGAGTCCAAACCTCTATTTGGAGACATTGACGGATC TACCTTCAAATCATATGCTGAAGCTAACATCCCTTTAGCCTACTATTTCTATGAGAACGAAGAACAACGTGCTGCTGCTGCCGATATTATTAAACCTTTTGCTAAAGAGCAACGTGGCAAAATTAACTTTGTTGGCTTAGATGCCGTTAAATTCGGTAAGCATGCCAAGAACTTAAACATGGATGAAGAGAAACTCCCTCTATTTGTCATTCATGATTTGGTGAGCAACAAGAAGTTTGGAGTTCCTCAAGACCAAGAATTGACGAACAAAGATGTGACCGAGCTGATTGAGAAATTCATCGCAGGAGAGGCAGAACCAATTGTGAAATCAGAGCCAATTCCAGAAATTCAAGAAGAGAAAGTCTTCAAGCTAGTCGGAAAGGCCCACGATGAAGTTGTCTTCGATGAATCTAAAGATGTTCTAGTCAAGTACTACGCCCCTTGGTGTGGTCACTGTAAGAGAATGGCTCCTGCTTATGAGGAATTGGCTACTCTTTACGCCAATGATGAGGATGCCTCTTCAAAGGTTGTGATTGCAAAACTTGATCACACTTTGAACGATGTCGA T AACGTTGATATTCAAGGTTATCCTACTTTGATCCTTTATCCAGCTGGTGATAAATCCAATCCTCAACTGTATGATGGATCTCGTGACCTAGAATCATTGGCTGAGTTTGTAAAGGAGAGAGGAACCCACAAAGTGGATGCCCTAGCACTCAGACCAGTCGAGGAAGAAAAGGAAGCTGAAGAAGAAGCTGAAAGTGAGGCAGACGCTCACGACGAGCTTTAA * The underlined part is a synonymous mutation to remove the BamH I and Sal I sites The specific sequence of Ero1 is as follows (SEQ ID NO.5): ATGAGGATAGTAAGGAGCGTAGCTATCGCAATAGCCTGTCATTGTATAACAGCGTTAGCAAACCCTCAAATCCCTTTTGACGGCAACTACACCGAGATCATCGTGCCAGATACCGAAGTTAACATCGGACAGATTGTAGATATTAACCACGAAATAAAACCCAAACTGGTGGAACTGGTCAACACAGACTTCTTCAAATATTACAAATTAAACCTATGGAAACCATGTCCGTTTTGGAATGGTGATGAGGGATTCTGCAAGTATAAGGATTGCTCTGT T GACTTTATCACTGATTGGTCTCAGGTGCCTGATATCTGGCAACCAGACCAATTGGGTAAGCTTGGAGATAACACGGTACATAAGGATAAGGGCCAAGATGAAAATGAGCTGTCCTCAAATGATTATTGCGCTTTGGATAAAGACGACGATGAAGATTTAGTATATGTCAATTTGATTGATAACCCTGAAAGATTCACCGGTTATGGTGGTCAGCAATCTGAATCTATTTGGACTGCGGTCTATGATGAGAACTGTTTCCAGCCGAATGAAGGATCACAATTGGGTCAAGTTGAAGACCTCTGTTTGGAGAAACAGATCTTTTACCGATTGGTTTCTGGTTTGCATTCTAGTATCTCCACCCACCTCACAAACGAATATCTGAATTTGAAAAATGGAGCATACGAACCAAATTTGAAACAGTTCATGATCAAAGTTGGGTATTTTACTGAAAGAATTCAAAACTTACATCTCAATTATGTCCTTGTATTGAAGTCACTAATAAAGCTACAAGAATACAATGTTATCGACAATCTACCTCTCGATGACTCTTTGAAAGCTGGTCTTAGCGGTTTAATATCTCAAGGAGCACAGGGTATTAACCAGAG TTCTGATGATTATCTATTTAACGAGAAGGTTCTTTCAAAATGACCAAAATGATGATTTGAAAAATGAATTCCGTGACAAATTCCGCAACGTGACTAGATTAATGGATTGTGTCCATTGCGAGAGATGCAAATTATGGGGAAAATTGCAAACTACAGGGTACGGGACTGCATTG AAGATTCTATTTGATTTGAAGAATCCTAATGACTCCATCAATTTAAAGAGAGTTGAGTTAGTTGCTCTAGTCAACACATTCCATAGATTGTCCAAATCTGTTGAAAGCATTGAAAACTTTGAAAAACTATATAAGATTCAACCGCCAACGCAGGATCGTGCATCAGCGTCGTCCG AATCCTTAGGCCTTTTCGATAACGAAGATGAACAAAATCTCCTCAACTCGTTTTCGGTTGATCAGGCAGTCATTTCATCGAAAGAGGCACCAGAAGAAATCAAAAGCAAACCTGTTGGAAAAGCCGCATATAAACAAAACAGTTGTCCATCATTGGGTTCAAAATCTATCAAAGA AGCATTCCATGAAGAACTTCACGCATTTATTGATGCAATTGGATTTATATTGAACTCTTACAGGACTTTGCCCAAGCTGTTGTACACACTTTTCCTCGTTAAATCATCTGAATTATGGGACATTTTCATTGGCACTCAAAGGCACCGAGATACCACATATAGAGTAGACTTGTAA *The underlined part is a synonymous mutation that removes the Sal I and Sac I sites Example 2: Electroporation of pNb plasmid into Pichia pastoris Extract plasmid: Inoculate DH5α bacteria carrying plasmid pNb into 10 mL LB liquid medium (containing 25 µg / mL zeocin) for recovery. Cultivate at 37°C, 200 rpm with shaking until OD600 reaches 1.0-2.0 (preferably 1.5-1.8), and extract plasmid according to the procedure of plasmid extraction kit.

[0027] Plasmid linearization: Take the recombinant plasmid pNb and digest it with restriction endonuclease. The reaction system is as follows:

[0028] The enzyme digestion condition was 37°C water bath for 3 h. After the reaction, 1.0% Agrose electrophoresis was used for identification.

[0029] The remaining samples were purified and recovered using a DNA purification kit (eluted with 30 μL) and the concentration was measured. The purified samples were used for subsequent experiments or stored in a -20°C refrigerator.

[0030] Preparation of competent cells of Pichia pastoris: 1) Take a GS115 cryovial from a -80°C freezer, streak it, and invert and culture it at 30°C for 2-3 days; 2) Pick a single clone and culture it in a 250mL shake flask (other specifications of shake flasks can be used) containing 25mL YPD, at 30°C, 220rpm overnight; 3) Take 1mL (1% inoculation) of the overnight culture and inoculate it into a 500mL shake flask (other inoculation ratios and other specifications of shake flasks can be used) pre-added with 100mL of fresh culture medium, and culture it at 30°C, 220rpm until OD 600 =1.0~2.0; 4) collect cells by centrifugation at 1500g for 5 min at 4°C, and suspend the cells with 90mL pre-cooled sterile water; 5) collect cells by centrifugation at 1500g for 5 min at 4°C, and suspend the cells with 90mL pre-cooled sterile water; 6) collect cells by centrifugation at 1500g for 5 min at 4°C, and suspend the cells with 20mL pre-cooled 1M sorbitol; 7) collect cells by centrifugation at 1500g for 5 min at 4°C, and suspend the cells with 1mL pre-cooled 1M sorbitol; 8) aliquot into 100 μL portions per tube and store in a -80°C refrigerator (valid for one week).

[0031] Electroporation: 1) Clean the clean bench, remove the 0.2 cm electroporation cup from the anhydrous ethanol, dry it in the clean bench (at least 30 min in advance), cover the electroporation cup with a lid, and place it in an ice box for precooling; 2) Thaw 1 competent cell on ice; 3) Mix 10-20 μL of linearized DNA with 80-100 μL of Pichia competent cells and transfer to the precooled electroporation cup; 4) Place on ice for 5 min; 5) Turn on the electroporator for preheating and perform electroporation according to the Pichia parameters recommended by the device used (MicroPulser electroporator: Fungi, Pic mode); 6) Immediately add 1 mL of precooled 1 M sorbitol to the cup, transfer the contents to a sterilized EP tube, and culture at 30°C, 220 rpm for 1-2 h; 7) Divide into 100 μL aliquots and plate the cells on medium containing different concentrations of bleomycin (100, 300, 500, 1000, and 1500 μL). The cells were plated on YPD selective plates containing 500 mg / L and cultured at 30°C for 2-3 days until single colonies appeared.

[0032] Example 3 Screening of recombinant nanobody strains Two clones were picked from each YPD selection plate with different concentrations of bleomycin (100, 300, 500 and 1000 mg / L) and inoculated into a conical flask containing 20 mL YPD. After culturing at 30°C and 220 r / min for 20 h, the bacteria were preserved in glycerol. The selected strains were inoculated at an inoculum of 1.0% into conical flasks containing 50 mL YPD and cultured at 30°C and 220 r / min for 96 h. After centrifugation (conditions: 4°C, 12000 r / min, 10 min), the supernatant was separated from the bacterial precipitate, and the supernatant was analyzed for protein by SDS-PAGE gel electrophoresis. The strain with the highest expression level was selected as host 1 (and as control) for the following experiments. The results are shown in Tables 1, 2 and 3, corresponding to Figure 5 , Figure 6 and Figure 7 .

[0033] Table 1 OD600 determination results of host 1 strains with different copy numbers at different times

[0034] Table 2 SDS-PAGE results of supernatant samples of host 1 fermentation broth with different copy numbers

[0035] Table 3 SDS-PAGE results of supernatant samples of host 1 fermentation broth with different copy numbers

[0036] From Table 1, Figure 5 It can be seen that there is little difference in the growth of SF01-SF07, and there is a slight difference in the growth of SF08. Figure 6 , Figure 7 It can be seen that on the third day of fermentation, the protein expression level of GS115 / pGAP-Nb-Z1.0-2 was the highest, twice that of the other strains, and the expression levels of the other strains were not much different. Therefore, the GS115 / pGAP-Nb-Z1.0-2 strain was used as host 1 for the next step.

[0037] Example 4: Electroporation of auxiliary protein plasmid into Pichia pastoris As in Example 2, the synthetic plasmids pAP1 and pAP2 were linearized, respectively, and electroporated into the competent host 1, respectively, and then plated on MD plates and YPD plates containing 0.25 mg / mL geneticin to screen for obtaining host 2 and host 3; As in Example 2, the synthetic plasmid pAP2 was linearized, electroporated into the competent host 2, and plated on a YPD plate containing 0.25 mg / mL geneticin to screen and obtain host 4.

[0038] The preparation process of host 1, host 2, host 3 and host 4 is as follows Figure 1 shown.

[0039] Example 5 Shake flask screening of host 1, host 2, host 3, and host 4 Plates of host 2, host 3, and host 4 were obtained from Example 4. 2 to 3 clones were picked from each plate, as well as host 1 glycerol bacteria and an empty host, and inoculated into a conical flask containing 20 mL YPD. After culturing at 30°C and 220 r / min for 20 h, glycerol bacteria were maintained; the selected strains were inoculated into conical flasks containing 50 mL YPD at an inoculum of 1.0%, and cultured at 30°C and 220 r / min for 96 h. Centrifuge (conditions: 4°C, 12000 r / min, 10 min), separate the supernatant from the bacterial precipitate, and perform SDS-PAGE gel electrophoresis on the fermentation supernatant to analyze the nanobody production. The results are shown in Tables 4, 5, and 6, corresponding to Figure 8 , Fig. 9 and Fig.10 . Table 4 OD600 determination results of empty host, host 1, host 2, host 3, host 4 at different times

[0040] Table 5 SDS-PAGE results of fermentation supernatant samples of empty host, host 1, host 2, host 3, and host 4

[0041] Table 6 SDS-PAGE results of fermentation supernatant samples of empty host, host 1, host 2, host 3, and host 4

[0042] From Table 4, Figure 8 It can be seen that there is little difference in the growth of SF01-SF06 and SF08-SF11, and the growth of SF07 is slightly slower than the others. Fig. 9 , Fig.10 , Fig.11It can be seen that on the third day of fermentation, the protein expression level of host 3-2 GS115 / pGAPZ-Nb-Z1.0-2 / pAP2-2 strain (with pAP2 introduced) was the highest, which was three times the expression level of its starting strain (host 1 GS115 / pGAPZ-Nb-Z1.0); the expression level of host 2-2 GS115 / pGAPZ-Nb-Z1.0-2 / pAP1-2 strain (with pAP1 introduced) was twice the expression level of its starting strain (host 1 GS115 / pGAPZ-Nb-Z1.0), but the expression effect of host 4 GS115 / pGAPZ-Nb-Z1.0-2 / pAP1 / pAP2, which had both pAP1 and pAP2 introduced, was not good (the expression level was similar to that of the starting strain host 1). It is speculated that the reason may be that the number of clones screened was small, or that the number of introduced genes was too large, resulting in high expression pressure on Pichia pastoris.

[0043] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A recombinant host cell for producing nanobodies, characterized in that The recombinant host cell contains genes encoding nanobodies and auxiliary proteins; The auxiliary proteins are: i) transcriptional regulatory protein Hac1 and heavy chain binding protein Bip; and / or, ii) protein disulfide isomerase Pdi and endoplasmic reticulum redox protein Ero1; The coding genes of the nanobody and the auxiliary protein exist in the host cell in the form of an expression vector, or are integrated into the genome of the host cell.

2. The recombinant host cell according to claim 1, characterized in that The recombinant host cell contains a first expression vector, a second expression vector and / or a third expression vector; The first expression vector contains a first expression cassette for expressing Nanobodies; The second expression vector contains a second expression cassette for expressing the transcriptional regulatory protein Hac1 and a third expression cassette for expressing the heavy chain binding protein Bip; The third expression vector contains a fourth expression cassette for expressing protein disulfide isomerase Pdi and a fifth expression cassette for expressing endoplasmic reticulum redox protein Ero1.

3. The recombinant host cell according to claim 2, characterized in that The promoter of the first expression cassette, the second expression cassette, the third expression cassette, the fourth expression cassette or the fifth expression cassette is P Gap Promoter.

4. The recombinant host cell according to claim 1, characterized in that The coding gene of the nanobody has a nucleotide sequence as shown in SEQ ID NO.

1.

5. The recombinant host cell according to claim 1, characterized in that The gene encoding the transcriptional regulatory protein Hac1 and the gene encoding the heavy chain binding protein Bip have nucleotide sequences shown in SEQ ID NOs. 2-3, respectively.

6. The recombinant host cell according to claim 1, characterized in that The gene encoding protein disulfide isomerase Pdi and the gene encoding endoplasmic reticulum redox protein Ero1 have nucleotide sequences shown in SEQ ID NOs. 4-5, respectively.

7. The recombinant host cell according to claim 1, characterized in that The host cell is a bacterial cell, a yeast cell or a mammalian cell.

8. The recombinant host cell according to claim 7, characterized in that The host cell is a Pichia pastoris cell.

9. The recombinant host cell according to claim 8, characterized in that The basic vector of the expression vector is a vector suitable for Pichia pastoris cell expression.

10. A method for producing a nanobody, characterized in that The method comprises: constructing the recombinant host cell according to any one of claims 1 to 9; culturing the recombinant host cell; and isolating and obtaining the nanobody from the culture product.

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