阅读说明：本技术 Tbab人源单克隆抗体重组载体、重组抗体及其制备方法 (TBAB humanized monoclonal antibody recombinant vector, recombinant antibody and preparation method thereof ) 是由 张涛 刘江海 杨菁 于 2020-06-10 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种甲状腺疾病Graves’病分型抗体TBAB人源单克隆抗体重组载体、重组抗体及其制备方法。针对TBAB抗体稳定性差、产量低等缺陷,本发明提供了一种重组TBAB人源单克隆抗体重组载体、重组抗体及其制备方法。本发明重组抗体制备时采用了CHO表达系统,构建稳定表达的细胞株,稳定表达量≥210mg/L,同时该抗体活性高,完全满足科研和检测的需求,实现该分型抗体的国产化,有助于提高疾病检出率,应用前景广阔。(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.)

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.

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.