CN111690680A

CN111690680A - TBAB humanized monoclonal antibody recombinant vector, recombinant antibody and preparation method thereof

Info

Publication number: CN111690680A
Application number: CN202010520755.XA
Authority: CN
Inventors: 张涛; 刘江海; 杨菁
Original assignee: Chengdu Tongyichuang Biotechnology Co ltd
Current assignee: Chengdu Tongyichuang Biotechnology Co ltd
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2020-09-22

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant vector of a Graves' disease typing antibody TBAB human monoclonal antibody, a recombinant antibody and a preparation method thereof. Aiming at the defects of poor stability, low yield and the like of a TBAB antibody, the invention provides a recombinant TBAB humanized monoclonal antibody recombinant vector, a recombinant antibody and a preparation method thereof. The recombinant antibody is prepared by adopting a CHO expression system, a stably expressed cell strain is constructed, the stable expression quantity is more than or equal to 210mg/L, and meanwhile, the antibody has high activity, completely meets the requirements of scientific research and detection, realizes the localization of the typing antibody, is beneficial to improving the disease detection rate, and has wide application prospect.

Description

TBAB humanized monoclonal antibody recombinant vector, recombinant antibody and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant vector of a Graves' disease typing antibody TBAB human monoclonal antibody, a recombinant antibody and a preparation method thereof.

Background

Autoimmune thyroid diseases (AITDs) are a group of Autoimmune diseases caused by the body's Autoimmune response to thyroid tissue. It is mainly composed of two diseases: graves 'disease (GD) and Hashimoto's Thyroiditis (HT). The common feature of AITD is the presence of multiple autoantibodies to thyroid tissue in the serum of patients, including thyrotropin receptor antibodies (TRAb), thyroglobulin antibodies (TGAb), thyroid peroxidase antibodies (TPOAb), and the like. In HT patients, the prevalence of TGAb is 25-50%, the prevalence of TPOAb is 90%, and the prevalence of TBAb (tbhr blocking antibodies), a member of the TRAb classification, is 17%. In the serum of GD patients, TRAb is increased by over 90%, theoretically, TRAb should be 100% positive, and the theory that TRAb is the main pathogenic factor of GD is met. In practice, however, TRAb is not 100% positive due to the sensitivity of the detection method. Trabs are a heterogeneous group of antibodies that can be further classified as thyroid stimulating antibodies (tsabs), thyroid blocking antibodies (tbabs) and neutral antibodies, depending on their respective effects on the TSHR. These antibodies are produced as a result of the immune system's loss of tolerance to thyroid antigens, a mechanism currently believed to be the result of multifactorial effects, including genetic susceptibility and environmental factors. The exact binding site of TSHR antibodies is described as a leucine rich region of the TSHR subunit. This is also the site of Thyroid Stimulating Hormone (TSH) binding. The stimulatory form of TRAb results in the same downstream effects as TSH and TSHR binding, including activation of adenylate cyclase leading to cyclic Adenosine Monophosphate Production (AMP), promoting fibroblast hyaluronan synthesis and activation of downstream signaling pathways.

At present, no TBAB typing antibody detection kit exists in the market, and meanwhile, a plurality of scholars are actively researching the effect of a TBAB antibody on Graves 'diseases and the action mechanism of the TBAB antibody, but the typing antibody TBAB has poor stability and low yield, needs to be purchased from abroad, has high cost and is inconvenient, so that the research on the relevant aspects of the effect of the TBAB antibody on the Graves' diseases is seriously hindered, and the technical progress is influenced.

Disclosure of Invention

Aiming at the defects of poor stability, low yield and the like of the TBAB antibody, the invention provides the recombinant TBAB humanized monoclonal antibody recombinant vector, the recombinant antibody and the preparation method thereof, so that the mass production of the antibody can be realized, and the diagnosis rate of the disease can be improved.

The invention provides a recombinant vector of a TBAB human monoclonal antibody, which comprises an expression frame for expressing the TBAB human monoclonal antibody, and the recombinant vector is constructed by inserting the expression frame into an expression vector.

In the recombinant vector of the TBAB human monoclonal antibody, the TBAB human monoclonal antibody comprises a heavy chain VH and a light chain VL.

Furthermore, the coding nucleotide sequence of the heavy chain VH is shown as SEQ ID NO. 1, and the coding nucleotide sequence of the light chain VL is shown as SEQ ID NO. 2.

Nucleotide sequence encoding the heavy chain VH of SEQ ID NO. 1

gacgtccagatccagcagcctgggactgagcttgtgaagcctggggcttcagtgagactgtcctgcaaggcttctggctacaccttcaccacctactggatgcactgggtgaagcagaggcctggacaaggccttgagtggatcggagagattgatccttctgatagttatactaactataatcaaaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcacctcagcagcctgacatctgaggactctgcggtctattactgttcaagaaactacggtagtggctactactttgactactggggccaaggcaccactctcacagtctcctca。

Nucleotide sequence encoding light chain VL of SEQ ID NO 2

ggcgttgagatgacacagtcgccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggagactgaagatgctgccacttattactgccagcagtggagtagtaacccgtggacgttcggtggaggcaccaaactggaaatcaaa。

In the recombinant vector of the TBAB human monoclonal antibody, the expression vector is pCaHO1.0.

The invention also provides a recombinant TBAB human monoclonal antibody, which is obtained by introducing a recombinant vector of the TBAB human monoclonal antibody into a host cell for expression and then purifying.

Wherein, the host cell is a CHO cell strain. The cell line was purchased from ATCC, ATCC CCL-61.

The invention also provides a preparation method of the recombinant TBAB human monoclonal antibody, which comprises the following steps:

a. constructing a TBAB human monoclonal antibody recombinant vector;

b. b, introducing the recombinant vector obtained in the step a into a receptor cell CHO by adopting an electrotransfer method, and screening a stable expression cell strain by puromycin with the final concentration of 10 mu g/mL and methotrexate with the final concentration of 200 nM/L;

c. and (3) culturing the stably expressed cell strain in a CD011 complete culture medium for 14 days, stopping culturing when the cell survival rate is less than 75%, collecting culture supernatant, and performing protein affinity purification on the supernatant to obtain the recombinant TSAB human monoclonal antibody.

Wherein the composition of the CD011 complete medium in the step c comprises: CD011 serum-free medium containing L-glutamine at a concentration of 1 mM/L.

Further, the CD011 serum-free medium is purchased from 88011-317, Jianshu science and technology Limited, Gansu; the L-glutamine was purchased from Sigma-Aldrich, G7513.

Wherein the glucose concentration is kept constant at 3-8g/L during the culture in the step c.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a recombinant TBAB human monoclonal antibody recombinant vector, a recombinant antibody and a preparation method thereof, wherein a reasonable recombinant vector is constructed, a CHO expression system is adopted to construct a stable expression cell strain, the stable expression quantity is more than or equal to 210mg/L, and meanwhile, the antibody has high activity, completely meets the requirements of scientific research and detection, realizes the localization of the typing antibody, is beneficial to improving the disease detection rate, and provides help for the treatment of diseases. The invention can simply and conveniently obtain a large amount of recombinant TBAB humanized monoclonal antibodies, solves the requirements of domestic scientific research work and rapid detection, and has wide application prospect.

Drawings

FIG. 1 is a graph showing the change in concentration of TBAB antibody protein with time in example 3;

FIG. 2 shows the SDS-PAGE detection of TBAB antibody protein molecular weight in example 4;

FIG. 3 shows the activity of thyroid epithelial cells cAM after stimulation with different concentrations of TBAB as in example 5.

Detailed Description

The present invention is further described in the following description of the embodiments with reference to the drawings, but the present invention is not limited thereto, and those skilled in the art can make various modifications or improvements based on the basic idea of the present invention within the scope of the present invention without departing from the basic idea of the present invention.

Each reagent used in the examples was a common commercially available product.

EXAMPLE 1 construction of pCHO1.0-TBAB expression plasmid

And constructing a stable expression cell strain.

The method comprises the following steps:

1. separating the plasma cells: collecting blood of Graves' disease patients, separating plasma cells by adopting a density gradient centrifugation method, extracting RNA, and obtaining cDNA by adopting an RT-PCR experimental technology.

2. Shown as SEQ ID NO. 1 and SEQ ID NO. 2.

3. The heavy chain variable region (VH) and light chain variable region (VL) of the hybridoma cells were PCR cloned using specifically designed upstream and downstream primers.

Wherein the heavy chain (VH) primer sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4:

an upstream primer: 5'cctaggcaaatgcagctggtgcag3'(SEQ ID NO:3)；

A downstream primer: 5'gtatactgaggagacggtgaccag 3'(SEQ ID NO:4)。

The light chain (VL) primer sequences are shown in SEQ ID NO:5 and SEQ ID NO: 6: :

an upstream primer: 5'gatatcctgcctgtgctgactcag 3'(SEQ ID NO:5)；

A downstream primer: 5'ttaattaactgcctgtgctgactcag 3'(SEQ ID NO:6)。

(1) Treating the heavy chain with endonuclease AvrII and BstZ17, treating the light chain with enzyme digestion by EcoRV and PacI, connecting the treated heavy chain and light chain with a cloning vector (pCHO1.0), transforming the connection product into competent bacteria DH5a, coating the transformed bacteria liquid on a Kana-resistant LB solid culture medium due to the pCHO1.0 vector with Kana + resistance genes, and culturing at 37 ℃ overnight;

the coding nucleotide of the endonuclease is shown as SEQ ID NO 7-10.

AvrII：cctagg(SEQ ID NO:7)；BstZ17：gtatac(SEQ ID NO:8)；EcoRV：gatatc(SEQID NO:9)；PacI：ttaattaa(SEQ ID NO:10)。

(2) The method comprises the following steps of (1) growing dispersed colonies by bacteria to be plated, selecting colonies with clear edges and good growth, and further sequencing and identifying;

(3) reserving candidate recombinant plasmids according to a sequencing result, performing PCR amplification again to obtain heavy chain (VH) and light chain (VL) sequences matched with an expression vector, connecting a PCR product with a linear expression vector (pCHO1.0) subjected to double enzyme digestion pretreatment, and transforming a competent bacterium DH5a by using a connecting product, wherein a transformed bacterium liquid can be coated on a Kana-resistant LB solid culture medium due to the fact that the expression vector carries a kanamycin (Kana +) resistance gene and is cultured at 37 ℃ overnight;

(4) sequencing method reference (2); comparing the two sequencing results, selecting the transformation bacteria with the correct sequence, and performing plasmid extraction after amplification culture.

EXAMPLE 2 introduction of pCHO1.0-TBAB expression plasmid into recipient cells to obtain recombinant antibody

The specific operation steps are as follows:

(1) co-transfecting the expression vector connected with the target monoclonal antibody heavy chain (VH) and light chain (VL) genes obtained in the example 1 into a eukaryotic expression cell strain CHO;

wherein, in the step (1), the eukaryotic expression cell strain CHO is cultured in a suspension culture and a CD011 complete culture medium under the culture conditions of 37 ℃ and 5 percent CO₂120rpm, counting CHO cells with trypan blue at a cell viability of above 98% at 0.5-1 × 10⁶Cells were seeded in CD011 complete medium at 37 ℃ in 5% CO at 120rmp₂And (5) culturing.

(2) Inoculation ofCounting the cell density when the day is marked as day 0, about day3 and day4, and if the cell density is more than 4 × 10⁶To the culture medium, 1mM sodium butyrate (obtained from Biotechnology, Inc., Shanghai) was added to the medium at a final concentration of 1mL, and fed CD feed 002 (obtained from 99014-008, Jianshun, Gansu) was added at a concentration of 5% of the culture volume, while the glucose concentration was controlled to be 3-8 g/L. Cells were transferred to 32 ℃ at 120rmp, 5% CO₂And (5) culturing.

(3) Thereafter, the glucose concentration was measured every other day, controlled at 3-8g/L and supplemented with 5% CD feed 002 at day4, 6, 8, 10.

(4) Detecting the survival rate of the cells, stopping culturing when the survival rate of the cells is lower than 75%, and collecting the culture solution. Harvesting supernatant after 7 days of continuous culture, centrifuging for 30min at 4000g, removing impurities such as cells in the supernatant, and filtering and sterilizing by using a 0.46um filter;

(5) the obtained supernatant contains the target antibody, and the high-purity antibody protein is obtained through conventional protein affinity purification and separation.

Example 3 determination of the content of recombinant antibody proteins

Every 1 day, 1mL of the medium was taken, and the antibody protein expression level was measured by BCA quantification method.

The method comprises the following specific steps:

(1) preparation of protein standards

a. 0.8mL of the protein standard preparation solution was added to a tube of protein standard (20mg BSA), and was dissolved sufficiently to prepare a 25mg/mL protein standard solution. Can be used immediately after preparation, or stored at-20 deg.C for a long time.

b. An appropriate amount of 25mg/mL protein standard was taken and diluted with medium to a final concentration of 0.5 mg/mL.

(2) Preparation of BCA working solution

Based on the number of samples, 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1) were prepared;

(3) protein concentration determination

a. Adding standard substance into standard substance well of 96-well plate at 0, 1, 2, 4, 8, 12, 16, 20 μ L, adding standard substance diluent to make up to 20 μ L, wherein the concentrations of standard substance are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL respectively.

b. Add 10. mu.L of sample to the sample wells of a 96-well plate. Add standard dilution to make up to 20 μ L.

c. 200. mu.l of BCA working solution was added to each well, and the mixture was left at 37 ℃ for 20 to 30 minutes.

d. And (3) measuring the absorbance of A562 or other wavelengths between 540 nm and 595nm by using a microplate reader.

e. The protein concentration of the sample was calculated from the standard curve and the sample volume used.

After 14 days of continuous detection, the content of the active ingredients is 0.5 to 1 × 10⁶The cells were seeded in CD011 complete medium at a concentration of/mL, with cell activity less than 75% at 14 days and antibody protein concentrations as high as 210mg/L (shown in FIG. 1).

Example 4 molecular weight determination of recombinant antibody proteins

The antibody protein was separated by SDS-PAGE electrophoresis and its molecular weight was determined. The specific operation steps are as follows:

(1) and preparing 12% separation gel.

(2) Pouring separation glue, keeping the glue surface 2cm away from the top surface, adding RO water seal, pouring the RO water after the glue is solidified in a gel manner at room temperature, sucking the RO water by using filter paper, pouring 5% concentrated glue, inserting a comb, and standing the mixture until the glue is solidified in a gel manner.

(3) Taking 50 mu L of protein sample, adding 15 mu L of 5 XLodding Buffer, boiling for 10min, centrifuging at 13000rpm for 10min, taking supernatant and adding into a sample tank.

(4) SDS-PAGE was performed (80V/30min,120V/70 min).

(5) When the bromophenol blue indicator ran to the bottom position of the separation gel, the electrophoresis was terminated.

(6) And placing the gel containing the target fragment in an R250 staining solution for shaking and staining for 30 min.

(7) And (5) placing the glue into a decoloring solution, shaking for decoloring, and taking out for photographing.

The experimental results show that the molecular weight of the antibody protein is about 25kd and 50kd according to SDS-PAGE electrophoresis, and the molecular weight is consistent with the theory (shown in figure 2).

Example 5 Activity assay for recombinant antibody proteins

Human normal thyroid epithelial cells were treated with TBAB antibody concentrations of 100ng/mL and 200ng/mL for 48h, and cAMP activity was measured using IgG purified from Graves' patient serum as a positive control. The specific operation is as follows:

(1) and (3) placing the cells treated by the antibody on ice, performing lysis treatment on the cells by a lysis solution for 30min, performing centrifugation treatment at 13000rpm for 15min, and taking and detecting a supernatant.

(2) In the test well plate, 100. mu.L/well of the standard or cell lysate was added and incubated at room temperature for 10 min.

(3) 25 μ L/well of HRP-cAMP working solution was added, placed on a shaker, and incubated at room temperature for 3 h.

(4) The liquid in the test plate was added with 200. mu.L/well of washing solution and washed 5 times.

(5) Add 100. mu.L/well of green fluorescent probe and incubate at room temperature in the dark for 3 h. OD was measured immediately with a microplate reader at a wavelength of 650 nm.

The results of the experiment show that cAMP activity is significantly reduced in thyroid epithelial cells after stimulation with TSAB antibody (as shown in FIG. 3).

The embodiment shows that the recombinant TSAB antibody protein can be efficiently prepared by the method, the expression level of the recombinant TSAB antibody protein is up to 210mg/mL, and the recombinant TSAB antibody protein has biological activity, can meet the requirements of scientific research work and clinical detection, and is beneficial to diagnosis and treatment of Graves' patients.

Sequence listing

<110> Chengdu and Tongxi Biotechnology Limited

<120> TBAB humanized monoclonal antibody recombinant vector, recombinant antibody and preparation method thereof

<141>2020-06-10

<160>10

<170>SIPOSequenceListing 1.0

<210>1

<211>357

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gacgtccaga tccagcagcc tgggactgag cttgtgaagc ctggggcttc agtgagactg 60

tcctgcaagg cttctggcta caccttcacc acctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagtta tactaactat 180

aatcaaaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcacctca gcagcctgac atctgaggac tctgcggtct attactgttc aagaaactac 300

ggtagtggct actactttga ctactggggc caaggcacca ctctcacagt ctcctca 357

<210>2

<211>318

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ggcgttgaga tgacacagtc gccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagtcaggc 120

acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 180

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggagactgaa 240

gatgctgcca cttattactg ccagcagtgg agtagtaacc cgtggacgtt cggtggaggc 300

accaaactgg aaatcaaa 318

<210>3

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

cctaggcaaa tgcagctggt gcag 24

<210>4

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gtatactgag gagacggtga ccag 24

<210>5

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gatatcctgc ctgtgctgactcag 24

<210>6

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ttaattaact gcctgtgctg actcag 26

<210>7

<211>6

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

cctagg 6

<210>8

<211>6

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gtatac 6

<210>9

<211>6

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gatatc 6

<210>10

<211>8

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ttaattaa 8

Claims

A recombinant vector for a TBAB human monoclonal antibody, characterized in that: the expression vector comprises an expression frame for expressing the TBAB human monoclonal antibody, and the expression frame is inserted into an expression vector to construct the expression vector.
2. The recombinant vector of a TBAB human monoclonal antibody of claim 1, characterized by: the TBAB human monoclonal antibody comprises a heavy chain VH and a light chain VL, wherein the coding nucleotide sequence of the heavy chain VH is shown as SEQ ID NO. 1, and the coding nucleotide sequence of the light chain VL is shown as SEQ ID NO. 2.
3. The recombinant vector of a TBAB human monoclonal antibody of claim 1, characterized by: the expression vector is pCaHO1.0.
4. A recombinant TBAB human monoclonal antibody characterized by: the TBAB human monoclonal antibody of any one of claims 1 to 3, which is purified after being introduced into a host cell and expressed.
5. The recombinant TBAB human monoclonal antibody of claim 4, wherein: the host cell is a CHO cell strain.
6. A host cell expressing the recombinant TBAB human monoclonal antibody of claim 4 or 5.
7. The method of producing a recombinant TBAB human monoclonal antibody of claim 4 or 5, comprising the steps of:

a. constructing a TBAB human monoclonal antibody recombinant vector;

b. b, introducing the recombinant vector obtained in the step a into a receptor cell CHO by adopting an electrotransfer method, and screening a stable expression cell strain by puromycin with the final concentration of 10 mu g/mL and methotrexate with the final concentration of 200 nM/L;

c. and (3) culturing the stably expressed cell strain in a CD011 complete culture medium for 14 days, stopping culturing when the cell survival rate is less than 75%, collecting culture supernatant, and performing protein affinity purification on the supernatant to obtain the recombinant TSAB human monoclonal antibody.
8. The method of claim 7, wherein the method comprises the steps of: the composition of the CD011 complete medium in the step c comprises the following steps: CD011 serum-free medium containing L-glutamine at a concentration of 1 mM/L.
9. The method of claim 7, wherein the method comprises the steps of: and c, keeping the glucose concentration constant at 3-8g/L during the culture.