Immune globulin light chain mutant, nucleotide, recombinant expression vector and application

Abstract

The invention relates to the technical field of bioengineering, in particular to an immunoglobulin light chain mutant, nucleotide, a recombinant expression vector and application. Escherichia coli is used as a chassis for heterologous expression of bovine immunoglobulin IgG light chains, an IgG light chain mutant with significantly improved soluble expression in Escherichia coli is obtained by using error-prone PCR and high-throughput screening, and a corresponding mutant strain is further constructed based on the IgG light chain mutant; wherein the soluble B-LC content of the mutant strain 2-8 (in an immune globulin light chain, the second amino acid is mutated into threonine from alanine, the 34 amino acid is mutated into aspartic acid from glycine, the 57 amino acid is mutated into lysine from glutamine, the 101 amino acid is mutated into valine from glutamic acid, and the 209 amino acid is mutated into arginine from tryptophan) is 29.1 mg / L; and the expression is 1.86 times that of the control soluble expression.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to immunoglobulin light chain mutants, nucleotides, recombinant expression vectors, and their uses. Background Technology

[0002] Immunoglobulins (Ig) are tetrameric proteins assembled from two light chains and two heavy chains. IgG is the most abundant immunoglobulin in serum, accounting for 80% of serum antibodies, with a molecular weight of 150 kDa (Kennedy PJ, Oliveira C, Granja PL, et al. Monoclonal antibodies: technologies for early discovery and engineering[J]. Critical Reviews in Biotechnology.2018, 38(3):394-408.). IgG is a symmetrical monomer composed of two identical light chains (LC) and two identical heavy chains (HC), with each chain linked by disulfide bonds (Durfee T, Nelson R, Baldwin S, et al. The complete genome sequence of Escherichia coli DH10B:insights into the biology of a laboratory workhorse[J]. Journal of Bacteriology. 2008, 190(7):2597-2606.). IgG is the most abundant antibody in the body, possessing antiviral, virus-neutralizing, antibacterial, and immunomodulatory functions (Damelang T, Brinkhaus M, van Osch TLJ, et al. Impact of structural modifications of IgG antibodies on effector functions[J]. Frontiers in Immunology. 2024, (8):1304365.). IgG is divided into variable regions (Fv) and constant regions according to the degree of change in its amino acid sequence. The variable regions include light chain variable regions (VL) and heavy chain variable regions (VH), which can recognize and bind to antigens. IgG is produced in mammalian cell systems (Lee YJ, Jeong KJ. Challenges to production of antibodies in bacteria and yeast[J]. Journal of Bioscience and Bioengineering, 2015, 120 (5): 483-490.), but it has limitations such as a long growth cycle and high investment costs.Therefore, shortening the growth cycle, reducing costs, increasing IgG expression levels, and obtaining high-throughput screening methods are urgent problems to be solved.

[0003] Escherichia coli is a Gram-negative bacterium and the most common host for recombinant protein production because it is genetically well-developed, can be used with commercial expression systems, is inexpensive to culture, and can achieve high cell densities in a short time (Lee YJ, Lee DH, Jeong KJ. Enhanced production of human full-length immunoglobulin G1 in the periplasm of Escherichia coli[J]. Applied Microbiology and Biotechnology, 2014, 98 (3): 1237-1246.). Besides secretory expression, protein expression is mainly localized in two places: the periplasmic space and the cytoplasm. The cytoplasm is a reducing environment, while the periplasmic space is an oxidizing environment. Because IgG contains disulfide bonds, the periplasmic space is the primary site for IgG expression (Billen B, Vincke C, Hansen R, et al. Cytoplasmic versus periplasmic expression of site-specifically and bioorthogonally functionalized nanobodies using expressed protein ligation[J]. Protein Expression and Purification, 2017, 133:25-34.). However, IgG expression in E. coli is mainly concentrated in inclusion bodies, with low levels of soluble protein expression.

[0004] Therefore, it is crucial to provide a technical solution that can solve the above-mentioned technical problems. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide immunoglobulin light chain mutants, nucleotides, recombinant expression vectors, and applications.

[0006] The objective of this invention can be achieved through the following technical solutions: The first objective of this invention is to provide an immunoglobulin light chain mutant, said immunoglobulin light chain mutant being selected from one of the following: (i) In the immunoglobulin light chain with the amino acid sequence shown in SEQ ID NO.1, the second amino acid is mutated from alanine to threonine, the third amino acid is mutated from glycine to aspartic acid, the fifth amino acid is mutated from glutamine to lysine, the tenth amino acid is mutated from glutamic acid to valine, and the twentieth amino acid is mutated from tryptophan to arginine. (ii) The amino acid sequence of the immunoglobulin light chain shown in SEQ ID NO.1 is such that the 104th amino acid is mutated from alanine to threonine; (iii) In the immunoglobulin light chain with the amino acid sequence shown in SEQ ID NO.1, the 5th amino acid is mutated from proline to serine, the 86th amino acid is mutated from lysine to isoleucine, and the 216th amino acid is mutated from cysteine ​​to serine; (iv) The amino acid sequence of the immunoglobulin light chain shown in SEQ ID NO.1 is such that amino acid 190 is mutated from glutamine to leucine; (v) In the immunoglobulin light chain with the amino acid sequence shown in SEQ ID NO.1, the 11th amino acid is mutated from valine to alanine, the 42nd amino acid is mutated from serine to threonine, the 96th amino acid is mutated from serine to threonine, and the 159th amino acid is mutated from isoleucine to valine.

[0007] Preferably, the immunoglobulin light chain mutant is as follows: In the immunoglobulin light chain with the amino acid sequence shown in SEQ ID NO.1, the second amino acid is mutated from alanine to threonine, the 34th amino acid is mutated from glycine to aspartic acid, the 57th amino acid is mutated from glutamine to lysine, the 101st amino acid is mutated from glutamic acid to valine, and the 209th amino acid is mutated from tryptophan to arginine.

[0008] A second objective of this invention is to provide nucleotides encoding the aforementioned immunoglobulin light chain mutant.

[0009] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned nucleotides.

[0010] A fourth objective of this invention is to provide a method for preparing a recombinant expression vector, comprising the following steps: (S1) The nucleotide sequence of bovine immunoglobulin light chain was optimized with E. coli codons and then site-directed mutagenesis was performed to obtain the immunoglobulin light chain mutant gene; (S2) Insert the immunoglobulin light chain mutant gene obtained in step (S1) into the basic plasmid to construct the recombinant expression vector.

[0011] In one embodiment of the present invention, in step (S1), the nucleotide sequence of the bovine immunoglobulin light chain optimized by E. coli codons is shown in SEQ ID NO.2.

[0012] In one embodiment of the present invention, in step (S2), the base plasmid is PET-30a(+) plasmid.

[0013] A fifth objective of this invention is to provide a host cell containing the above-described recombinant expression vector.

[0014] In one embodiment of the present invention, the host cell is Escherichia coli BL21(DE3).

[0015] A fifth objective of this invention is to provide the use of the above-mentioned immunoglobulin light chain mutants, nucleotides, recombinant expression vectors, or host cells in enhancing the soluble expression of immunoglobulin light chains.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention uses Escherichia coli as a chassis to heterologously express bovine immunoglobulin IgG light chains, and uses error-prone PCR and high-throughput screening to obtain IgG light chain mutants with significantly enhanced soluble expression in E. coli, and further constructs corresponding mutant strains based on this; thus laying the foundation for efficient IgG expression. Attached Figure Description

[0017] Figure 1 (a) is a diagram of the construction of the B-LC expression plasmid; (b) is a diagram of the construction of the PET-30a(+)-B-LC plasmid; M: Marker, lane 1 is the control PET-30a(+), lane 2 is the supernatant of the B-LC lysis buffer, and lane 3 is the precipitate of the B-LC lysis buffer.

[0018] Figure 2 This is a diagram showing the construction of error-prone PCR mutant libraries; M: 2000 bp Marker; lanes 1-16 represent the validation results of recombinant plasmid mutant libraries; Figure 3 (a) is the screening diagram of the error-prone PCR mutation library; (b) is the initial screening diagram of the error-prone PCR mutation library. Figure 4 (a) Sequence alignment and conservation analysis of the B-LC error-prone PCR mutant library; (b) Nucleic acid sequence alignment; Figure 5The graph shows the expression content of soluble protein in B-LC mutant; (a) is the SDS-PAGE analysis graph of soluble protein in B-LC mutant, M: Marker, lane 1 is WT, lane 2 is 3-3, lane 3 is 7-3, lane 4 is 7-11, lane 5 is 6-11, and lane 6 is 2-8; (B) is the graph showing the change in soluble protein content in B-LC mutant. Detailed Implementation

[0019] Because of its advantages such as clear genetic background, simple culture operation, and significantly high levels of exogenous gene products, *E. coli* has become the most widely used and important classical expression system. The periplasmic space of *E. coli* is an oxidative environment, which is conducive to disulfide bond formation. However, heterologously expressed IgG mainly exists in the form of inclusion bodies, resulting in low soluble expression levels. This makes it difficult to obtain easily purified and active heterologous proteins.

[0020] Current research mainly focuses on using E. coli redox mutants to express both the antigen-binding fragment (Fab) and full-length IgG of IgG. However, studies on improving soluble expression by mutating IgG amino acids are limited, and methods for screening IgG expression levels are complex, hindering the efficient utilization of immunoglobulins in practical production. Therefore, improving the soluble expression level of IgG in E. coli will provide a novel pathway for its efficient utilization.

[0021] This invention utilizes error-prone PCR and high-throughput screening to obtain IgG light chain mutants with significantly enhanced soluble expression in Escherichia coli, laying the foundation for efficient IgG expression.

[0022] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0023] In the following examples, the bovine IgG light chain amino acid sequence (AAK84156.1, abbreviated as "B-LC") was obtained from the NCBI database. The B-LC amino acid sequence (SEQ ID NO.1) is as follows: MAWSPLLLLTLVALCTGSWAQAVLTQPSSVSGSLGQRVSITCSGSSDNIGRYDVDWYQQVPGSGLRTILYIGSSRPSGVPDRFSGSKSGNTATLTISSLQAEDEADYFCATGDDSSRT AVFGSGTTLTVLGQPKSAPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWKADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS The sequence of the B-LC gene optimized for E. coli codons (SEQ ID NO.2) is as follows: atggcttggtcaccattattattaacacttgttgctttgtgcaccggttcatgggctcaagctgttttaacacaaccatcatcagtttcaggctcattaggccaacgtgtttcaattacatgctcaggttcatcagataacatcggtcgttacgacgttgactggtatcaacaagttccaggttcaggtttacgtacaattttatacattggttcatcacgtccaagcggtgttccagatcgtttttcaggttcaaaatcaggtaataccgctacacttacaatttcaagccttcaggctgaggatgaggctgactatttttgcgctacaggtgatgactcatcacgtacagctgtttttggttcaggtacaacattaacagtcttgggccaaccaaagtcagctccatcagttacattatttccaccgtcaacagaggagcttaatggtaacaaggctacacttgtttgtctcatctcagatttttatccgggttcagttacagttgtttggaaggctgatggttcaacaattacacgtaatgttgagacaacacgtgcctcaaaacagtcaaattcaaaatacgccgcttcatcataccttagccttacatcatcagactggaaatcaaagggttcatactcatgtgaagttacacacgagggttcaacagttacaaaaacagttaaaccatcagagtgcagc The sequence of primer B-LC-F (SEQ ID NO.3, 5'-3') is as follows: ctttaagaaggagatatacatatgaaatacttattaccaacagctgctg; The sequence of primer B-LC-R (SEQ ID NO.4, 5'-3') is as follows: catggtggccgaccggtggctgcactctgatggtttaact; The sequence of primer 30-RFP-F (SEQ ID NO.5, 5'-3') is as follows: caccggtcggccaccatggcgagtagcg; The sequence of primer 30-RFP-R (SEQ ID NO.6, 5'-3') is as follows: ggccatagctggttgagcagcaagtaataataaac; The specific components of the culture medium and buffer solution are as follows: (1) LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, used for culturing Escherichia coli; (2) LB solid medium: Add 20 g / L agar powder to LB liquid medium; (3) PBS buffer solution (10×): Na2HPO4 80 mM, NaCl 1.36 M, KH2PO4 20 mM, KCl 26 mM, pH 7.4; The specific instruments and equipment are as follows: Spectra Max i3x multi-functional microplate reader, Nanodrop 2000 ultra-micro nucleic acid and protein quantification instrument, S1000 PCR instrument, Power basic electrophoresis instrument, ChemiDoc XRS+ gel imaging system.

[0024] Unless otherwise specified, all reagents used are commercially available, and all detection methods and techniques used are conventional in this field.

[0025] Example 1 This embodiment provides a specific experimental procedure, as follows: (1) Preparation and transformation of competent Escherichia coli cells Preparation of competent states: picking E. coli Top 10 or E. coli A single colony of BL21 (DE3) was incubated in 4 mL LB medium on a shaker for 12 h (37°C, 200°C). rpm Then, inoculate 1% into 50 mL of LB medium and incubate on a shaker until OD (October Expiratory Time) is reached. 600 The concentration is 0.3~0.5, centrifuged (4℃, 4500). rpm Collect bacterial cells (10 min). Wash the bacterial cells with 0.1 mol / L CaCl2 solution, then resuspend the bacterial cells in 1 mL of 0.1 mol / L CaCl2 solution containing 15% glycerol. Aliquot the bacterial cells into 100 μL tubes and store at -80℃ for later use.

[0026] Transformation experiment: Competent cells were mixed with 10 μL of ligation product and incubated on ice for 30 min. After heat shock at 42℃ for 90 s, 900 μL of LB medium was added, and the cells were cultured on a shaker for 60 min (37℃, 200°C). rpm Centrifugation (4500) rpm Bacterial cells were collected (after 5 min) and plated on antibiotic-resistant plates, then incubated at 37°C to obtain clones. PCR amplification was used to verify the product size.

[0027] (2) Error-prone PCR amplification and mutant library construction PCR reaction system (20 μL): RandomMut Buffer (10×) 2 μL, Mutation enhancer (10×) 2 μL, dNTP (2.5mM each) 2 μL, DNA template 1 μL, primer mixture (10 μM each) 0.4 μL, RandomMut DNA polymerase 0.4 μL, ddH2O added to 20 μL.

[0028] PCR amplification program: 94℃ for 3 min; 94℃ for 10 s; 55℃ for 30 s; 72℃ for 1 min / kb, 30 cycles; 72℃ for 10 min.

[0029] PCR products Dpn I digested the sample and verified the amplified product by agarose gel electrophoresis.

[0030] PCR product recovery: The target fragment amplified into a single band was recovered using a gel extraction and recovery kit, and the DNA concentration after recovery was measured.

[0031] (3) Seamless cloning reaction system The target fragment and linearized vector amplified by PCR were recovered using a gel recovery kit, and the concentration of the recovered DNA was determined using a Nanodrop 2000 micro-volume nucleic acid and protein quantification instrument.

[0032] The ligation was completed according to the Clon Express II one-step cloning kit steps. The molar ratio of the vector to the target fragment was 1:2. The 10 μL reaction system included 0.4 pmol of the target fragment, 0.2 pmol of the vector fragment, 2 μL of 5×CEⅡ Buffer, 1 μL of Exnase, and ddH2O.

[0033] (4) Construction of heterologous expression plasmids Protein purification plasmid construction: Using the IgG light chain as a template, the target gene B-LC (as shown in SEQ ID NO.2) was amplified using primers B-LC-F and B-LC-R. After verifying the correct bands by agarose gel electrophoresis, the plasmid was purified and recovered. The target gene and PET-30a(+) plasmid were then purified using restriction enzymes. Nde I and Xho I. The target gene and the linearized vector were digested to obtain a linear vector; then the target gene and the linearized vector were seamlessly cloned and ligated, and the product was transformed into E. coli Top10 competent cells. The plasmid PET-30a(+)-B-LC was selected for resistance screening; then it was introduced into E. coli BL21(DE3) competent cells, and heterologous expression strains were obtained through resistance screening.

[0034] (5) Protein expression Plasmid PET-30a(+)-B-LC was transformed into competent Escherichia coli BL21(DE3) cells. Correct single colonies were picked and activated in 4 mL LB medium for 12 h. The colonies were then inoculated at a 3% ratio into LB liquid medium and cultured at 37°C with shaking until OD (October Expected). 600 The initial concentration was approximately 0.6–0.8. IPTG was added to a final concentration of 1 mmol / L, and induction was performed overnight at 37°C. Centrifugation was then performed at 4°C, 8000 rpm. rpm Collect bacterial cells (10 min), wash twice with PBS buffer and resuspend, then sonicate on ice (800 W, 5 s operation, 5 s pause). Centrifuge (4℃, 10000 rpm). rpm Separate the precipitate and supernatant (10 min). Take the original bacterial culture, the supernatant after sonication, and the precipitate (PBS resuspended) respectively, treat with sample loading buffer, and observe protein expression using 12.5% ​​SDS-PAGE.

[0035] (6) Determination of soluble protein content The soluble protein content was determined using the BCA method, as detailed below: The original bacterial culture was sonicated and centrifuged to collect the supernatant. 20 μL of the supernatant was accurately added to a 96-well plate. PBS was used as a blank control. 200 μL of BCA working solution was added to the sample wells. The plate was incubated at 37°C for 30 min and the absorbance was measured at 562 nm.

[0036] A standard curve was plotted with bovine serum albumin concentration on the x-axis and the corresponding absorbance on the y-axis, yielding the linear regression equation: y = 0.997x + 0.0123, R0. 2 =0.9996.

[0037] The content of soluble protein in the sample was then calculated based on the above linear regression equation.

[0038] (7) Data Analysis All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation. The experimental data were analyzed using SPSS 17.0 statistical software for variance analysis. p A value <0.05 was considered statistically significant; Origin 8.5 software was used for plotting.

[0039] Example 2 This embodiment provides the results and analysis of the experiment described in Embodiment 1, as follows: (1) Expression of immunoglobulin IgG light chain gene in Escherichia coli B-LC consists of 235 amino acids, totaling 705 bp. XhoI and NdeI restriction enzyme sites were added to the 5' and 3' ends of the codon-optimized B-LC nucleotide sequence, respectively, and then inserted into the PET-30a(+) expression plasmid, successfully constructing the B-LC E. coli expression vector: PET-30a(+)-B-LC plasmid. Figure 1 a. Synthesized by a contracted company. However, B-LC expression in E. coli is mainly concentrated in inclusion bodies, with low levels of soluble protein expression. Figure 1 (b) To improve this problem, B-LC was randomly mutated. To quickly determine the soluble expression status of B-LC after random mutation in *E. coli*, a rapid detection method was further developed to avoid the need for time-consuming SDS-PAGE observation of protein expression, as follows: In the constructed PET-30a(+)-B-LC expression plasmid, a red fluorescent protein reporter gene (RFP) is linked to the B-LC gene via a linker (SEQ ID NO.7, 5'-3': caccggtcggccacc) to obtain the PET-30a(+)-B-LC-RFP plasmid. The expression status of B-LC after random mutation can be qualitatively reflected by the fluorescence intensity of RFP.

[0040] (2) Construction of B-LC error-prone PCR mutant library Using plasmids containing PET-30a(+)-B-LC as templates, error-prone PCR amplification of B-LC was performed using the QuickMutation™ random gene mutation kit with primers B-LC-F and B-LC-R (Table 1).

[0041] Using the PET-30a(+)-B-LC-RFP plasmid as a template, the PET-30a(+)-RFP linear vector was obtained by reverse amplification using primers 30-RFP-F and 30-RFP-R. The PET-30a(+)-RFP linear vector (5997 bp) and a randomly mutated B-LC (705 bp) were ligated seamlessly. Colony PCR verification showed that the band size was consistent with that of 1803 bp. Figure 2 After successful sequencing, 500 colonies were collected to obtain the B-LC error-prone PCR mutant library.

[0042] (3) Screening of B-LC error-prone PCR mutant libraries High-throughput screening was performed using a 96-well plate and an ELISA reader. The B-LC error-prone PCR mutant library obtained in the initial screening step (2) yielded 30 mutant strains with higher fluorescence intensity than the control strain. Figure 3 a), and after further screening and verification, 6 positive mutant strains were obtained ( Figure 3 b), accounting for 20% of the total mutant library.

[0043] Five B-LC mutants with fluorescence values ​​increased by 32.3%–118.8% compared to the control strain (PET-30a(+)-B-LC strain) were further analyzed. Sequence alignment and conservation analysis of these five B-LC mutants were performed using Snap Gene software. Figure 4 As can be seen from a, the base mutation rate is 0.14%~0.71%, the mutation sites are evenly distributed, and there is no base bias. From Figure 4 b shows that five amino acids were mutated in B-LC of mutant strains 2-8: alanine (A) mutated to threonine (T), glycine (G) mutated to aspartic acid (D), glutamine (Q) mutated to lysine (K), glutamic acid (E) mutated to valine (V), and tryptophan (W) mutated to arginine (R). Among them, three hydrophobic amino acids were changed to one, suggesting that the mutation of amino acids from hydrophobic to hydrophilic may have a significant impact on improving the soluble expression of B-LC.

[0044] (4) The B-LC mutant is solublely expressed in Escherichia coli. Five B-LC mutants with increased RFP fluorescence intensity were finally obtained through secondary screening: 3-3 (the second amino acid in the immunoglobulin light chain was mutated from alanine to threonine, the third amino acid from glycine to aspartic acid, the fifth amino acid from glutamine to lysine, the tenth amino acid from glutamic acid to valine, and the twentieth amino acid from tryptophan to arginine), 7-3 (the eleventh amino acid in the immunoglobulin light chain was mutated from valine to alanine, the thiron amino acid from serine to threonine, the 96th amino acid from serine to threonine, and the twentieth amino acid from isoleucine to valine), 7-11 (the 190th amino acid in the immunoglobulin light chain was mutated from glutamine to leucine), and 6-11 (the fifth amino acid in the immunoglobulin light chain was mutated from proline to leucine). The following amino acids were modified: 1) serine (replaced from lysine to isoleucine at position 86, cysteine ​​to serine at position 216), 2-8 amino acids (replaced from alanine to threonine at position 2 of the immunoglobulin light chain, from glycine to aspartic acid at position 34, from glutamine to lysine at position 57, from glutamic acid to valine at position 101, and from tryptophan to arginine at position 209), and 2) were constructed into the PET-30a(+) vector (inserted between the XhoI and NdeI restriction enzyme sites). These were then transformed into *E. coli* to obtain the corresponding engineered strains. Further analysis of soluble expression levels was performed, using *E. coli* containing the PET-30a(+)-B-LC plasmid as a control. All mutant strains showed a band at 25 kDa, indicating successful expression of the B-LC mutant. Figure 5 a). The soluble B-LC content of mutant strain 2-8 was 29.1 mg / L, which was 1.86 times that of the control soluble expression (15.6 mg / L), indicating that the B-LC mutant 2-8 increased the soluble expression of B-LC protein. Figure 5 ).

[0045] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. An immunoglobulin light chain mutant, characterized in that, The immunoglobulin light chain mutant is selected from one of the following: (i) In the immunoglobulin light chain with the amino acid sequence shown in SEQ ID NO.1, the second amino acid is mutated from alanine to threonine, the third amino acid is mutated from glycine to aspartic acid, the fifth amino acid is mutated from glutamine to lysine, the tenth amino acid is mutated from glutamic acid to valine, and the twentieth amino acid is mutated from tryptophan to arginine. (ii) The amino acid sequence of the immunoglobulin light chain shown in SEQ ID NO.1 is such that the 104th amino acid is mutated from alanine to threonine; (iii) In the immunoglobulin light chain with the amino acid sequence shown in SEQ ID NO.1, the 5th amino acid is mutated from proline to serine, the 86th amino acid is mutated from lysine to isoleucine, and the 216th amino acid is mutated from cysteine ​​to serine; (iv) The amino acid sequence of the immunoglobulin light chain shown in SEQ ID NO.1 is such that amino acid 190 is mutated from glutamine to leucine; (v) In the immunoglobulin light chain with the amino acid sequence shown in SEQ ID NO.1, the 11th amino acid is mutated from valine to alanine, the 42nd amino acid is mutated from serine to threonine, the 96th amino acid is mutated from serine to threonine, and the 159th amino acid is mutated from isoleucine to valine.

2. The immunoglobulin light chain mutant according to claim 1, characterized in that, The specific immunoglobulin light chain mutants are as follows: In the immunoglobulin light chain with the amino acid sequence shown in SEQ ID NO.1, the second amino acid is mutated from alanine to threonine, the 34th amino acid is mutated from glycine to aspartic acid, the 57th amino acid is mutated from glutamine to lysine, the 101st amino acid is mutated from glutamic acid to valine, and the 209th amino acid is mutated from tryptophan to arginine.

3. Nucleotides encoding the immunoglobulin light chain mutant of claim 1.

4. A recombinant expression vector containing the nucleotides as described in claim 3.

5. A method for preparing the recombinant expression vector as described in claim 4, characterized in that, Includes the following steps: (S1) The nucleotide sequence of bovine immunoglobulin light chain was optimized with E. coli codons and then site-directed mutagenesis was performed to obtain the immunoglobulin light chain mutant gene; (S2) Insert the immunoglobulin light chain mutant gene obtained in step (S1) into the basic plasmid to construct the recombinant expression vector.

6. The method for preparing a recombinant expression vector according to claim 5, characterized in that, In step (S1), the nucleotide sequence of the bovine immunoglobulin light chain after E. coli codon optimization is shown in SEQ ID NO.

2.

7. The method for preparing a recombinant expression vector according to claim 5, characterized in that, In step (S2), the base plasmid is PET-30a(+) plasmid.

8. A host cell containing the recombinant expression vector of claim 5.

9. The host cell according to claim 8, characterized in that, The host cell was Escherichia coli BL21(DE3).

10. The use of the immunoglobulin light chain mutant as described in claim 1, the nucleotide as described in claim 3, the recombinant expression vector as described in claim 4, or the host cell as described in claim 8 in enhancing the soluble expression of immunoglobulin light chains.

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