CN116655791B

CN116655791B - Nanometer antibody for resisting prolactin receptor, recombinant vector, recombinant bacterium and application

Info

Publication number: CN116655791B
Application number: CN202310508845.0A
Authority: CN
Inventors: 赵伟军; 白可可; 陈雪丹; 姜玲
Original assignee: Zhejiang Contact Biotechnology Co ltd
Current assignee: Zhejiang Contact Biotechnology Co ltd
Priority date: 2023-05-06
Filing date: 2023-05-06
Publication date: 2023-11-07
Anticipated expiration: 2043-05-06
Also published as: CN116655791A

Abstract

The invention provides a nano antibody for resisting a prolactin receptor, a recombinant vector, recombinant bacteria and application, and belongs to the technical field of nano antibodies. The invention screens phage display library to obtain nano antibody capable of binding with high affinity with prolactin receptor, and the amino acid sequence is shown as SEQ ID NO. 1. Experiments show that 30 nano antibodies CQ01-CQ30 are screened, and the affinity test shows that the nano antibody CQ24 protected by the invention has the highest affinity with human prolactin receptor-ECD protein. The CQ24 nano antibody obtained by screening can be used for preparing medicaments for inhibiting the prolactin receptor, and provides a new means for treating diseases caused by the abnormality of the prolactin and the prolactin receptor.

Description

Nanometer antibody for resisting prolactin receptor, recombinant vector, recombinant bacterium and application

Technical Field

The invention belongs to the technical field of nanobodies, and particularly relates to a nanobody for resisting a prolactin receptor, a recombinant vector, recombinant bacteria and application.

Background

Prolactin is a polypeptide hormone extracted from bovine pituitary glands in 1477 years and is named because it can promote lactation in rabbits. In the human genome, the gene encoding prolactin is located on chromosome 6. The prolactin gene is 10kb in length and consists of 5 exons and 4 introns. Prolactin is secreted by the pituitary gland pituitary region, and many sites external to the pituitary gland, principally including lymphocytes, epithelial cells of the lactation breast, epithelial-derived breast cancer cells, and placenta. The manner in which prolactin acts is three: autocrine, paracrine and endocrine.

Prolactin receptors are widely found in peripheral organs such as the pituitary, heart, lung, brain, thymus, spleen, liver, pancreas, kidney, adrenal gland, uterus, skeletal muscle and skin. Prolactin receptors can be largely divided into extracellular domains, transmembrane domains and intracellular activation signaling domains. Prolactin binds to the extracellular domain of the prolactin receptor, activates downstream signal transduction pathways, and modulates several physiological and biochemical processes, such as those involved in breast development, lactation, angiogenesis, preeclampsia, tumors, maternal diabetes and anti-apoptotic effects.

Downstream signaling pathways for prolactin receptors are primarily Jak1/Stat3, jak2/Stat5, PI3K/Akt, and Raf/MEK/ERK signaling pathways. STAT proteins comprise a DNA binding domain, an SH 3-like domain, an SH 2-like domain, an NH 2-and a COOH-terminal activation domain. Phosphorylated prolactin receptors interact with SH2 domains of STAT. When STAT interacts with the prolactin receptor, it is phosphorylated by receptor-associated Jak kinases. The phosphorylated STAT molecule is separated from the receptor, and the phosphorylated tyrosine residue thereof is combined with the SH2 domain of another phosphorylated STAT molecule to form exclusive or homodimerization. Finally, STAT dimers are transferred to the nucleus, activating STAT DNA binding motifs in the target gene promoters.

Studies have shown that prolactin and prolactin receptor abnormalities are associated with a variety of cancers, and that inhibition of the downstream signaling pathway of prolactin can be used to treat cancer cell growth both in vitro and in vivo experiments. But existing patents and techniques cannot convert them to therapeutic applications. Thus, there is a need for antibodies with high affinity, more stable, and unlimited modes of administration for the treatment of prolactin and diseases caused by abnormal prolactin receptors.

Nanobody (Nb) is a naturally deleted light chain antibody found in the peripheral blood of alpaca in 1989. Nanobodies, which contain only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as heavy chain single domain antibodies, due to the deletion of the light chain, have a molecular weight (about 15 kDa) of about one tenth of that of conventional antibodies, with a protein crystal structure length of 4nm and a diameter of 2.5nm. Is the smallest fragment found at the present stage that can bind to antigen. The nano antibody has good tissue penetrability due to small volume and molecular weight, can penetrate through the blood brain barrier to reach focus tissues of the traditional antibody to feed, is discharged, has short half-life period and greatly avoids toxic effects. Therefore, the nanobody can be applied to inhibiting the combination of the prolactin and the prolactin receptor to effectively avoid the activation of a downstream signal transduction pathway, thereby treating diseases caused by the abnormality of the prolactin/the prolactin receptor. However, nanobodies with high affinity for the prolactin receptor are currently lacking.

Disclosure of Invention

In view of the above, the present invention aims to provide an anti-prolactin receptor nanobody which has the characteristic of high affinity binding with the prolactin receptor.

The invention also aims to provide a recombinant vector and recombinant bacteria for expressing the nano antibody, and provides rich materials for treating diseases caused by abnormal prolactin receptors.

The invention provides a nano antibody of an anti-prolactin receptor, and the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1.

The invention provides a nano antibody fusion protein of an anti-prolactin receptor, which is a signal peptide-connecting peptide-the nano antibody-Fc region, and the amino acid sequence is shown as SEQ ID NO. 2.

The invention provides a coding gene of the nano antibody fusion protein, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 3.

The invention provides a recombinant expression vector containing the coding gene.

Preferably, the backbone vector of the recombinant expression vector is pcDNA3.1.

Preferably, the multiple cloning site of the coding gene inserted into the framework vector is Not1/Xba1.

The invention provides engineering bacteria containing the recombinant expression vector.

Preferably, the host bacteria of the engineering bacteria comprise a prokaryotic expression system and/or a eukaryotic expression system.

The invention provides application of the nanobody, the nanobody fusion protein, the coding gene, the recombinant expression vector or the engineering bacterium in preparing medicaments for inhibiting the combination of prolactin and a prolactin receptor.

The invention provides application of the nanobody, the nanobody fusion protein, the coding gene, the recombinant expression vector or the engineering bacterium in preparing medicines for preventing and/or treating diseases caused by abnormal prolactin or prolactin receptor;

preferably, the disease comprises at least one of: infertility, galactorrhea, prolactinoma, amenorrhea, menoxenia, vision disorders, hypopituitarism, systemic lupus erythematosus, systemic scleroderma, behcet's disease, drying syndrome, psoriasis, hypercarrheaemia, acne and breast cancer.

The invention provides a nano antibody of an anti-prolactin receptor, and the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1. The invention screens and obtains the nano antibody which can combine with the prolactin receptor with high affinity by utilizing phage display library. Experiments show that 30 nano antibodies CQ01-CQ30 are screened out, and the affinity test shows that the EC of the nano antibody CQ24 protected by the invention when combined with human prolactin receptor-ECD protein ₅₀ EC of 4.6ng/mL, but other kinds of nanobodies ₅₀ Are all larger than 4.6ng/mL and are far higher than the nano antibody CQ24. Therefore, the CQ24 nano antibody obtained by screening of the invention binds with high affinity to the prolactin receptor, can be used for preparing medicaments for inhibiting the prolactin receptor, and provides a new means for treating diseases caused by the abnormality of the prolactin and the prolactin receptor.

Drawings

FIG. 1 shows the results of affinity of CQ24 nanobodies with human prolactin receptor-ECD conjugates;

FIG. 2 shows the phosphorylation inhibition results of STAT5 by CQ24 nanobody at various concentrations;

FIG. 3 is a graph comparing the phosphorylation inhibition of STAT5 by different nanobodies;

FIG. 4 shows the results of affinity of conventional antibodies to human prolactin receptor-ECD conjugates.

Detailed Description

The invention provides an anti-prolactin receptor nano-antibody, the amino acid sequence of which is shown as SEQ ID NO. 1 (QVQLVESGGGSVQAGGSLRLSCVASGYTRRGNCLG WFRQAPGKEREGVASIYASSGWTSYADSVKGRFTISLDNAKTMVYLQMN SLNPEDTAMYFCAAADTGLCGLGARIYSYWGQGTQVTVSS).

In the invention, the nanobody has the characteristic of high affinity binding to the prolactin receptor, and has higher affinity binding advantage compared with other nanobodies screened in phage surface display libraries constructed in the same batch.

The invention provides a nano antibody fusion protein CQ24-Fc of an anti-prolactin receptor, which is a signal peptide-connecting peptide-the nano antibody-Fc region, and the amino acid sequence is shown as SEQ ID NO. 2 (METDTLLLWVLLLWVPGSTGDSQVQLVESGGGSVQAGGSLRLSCVASGYTRRGNCLGWFRQAPGKEREGVASIYASSGWTSYADSVKGRFTISLDNAKTMVYLQMNSLNPEDTAMYFCAAADTGLCGLGARIYSYWGQGTQVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLGK). Wherein the amino acid sequence of the signal peptide is shown as SEQ ID NO. 4 (METDTLLLWVLLLWVPGSTGD). The connecting peptide is S. The amino acid sequence of the Fc region is preferably SEQ ID NO:5 (DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLGK).

The invention provides a coding gene of the nano antibody fusion protein, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 3 (atggagaccgatacactgctgctgtgggtgctgctgctgtgggtgccaggctccaccggagattcc)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGTAG CCTCTGGATACACCAGGCGTGGAAATTGCTTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGC AAGTATTTATGCGAGTAGTGGTTGGACATCCTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCTAGACAAC GCCAAGACCATGGTATATCTACAAATGAACAGCCTGAACCCTGAGGATACTGCCATGTACTTCTGTGCAGCAGCGG ATACGGGACTTTGTGGCTTGGGTGCGAGGATCTATAGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA Wherein the lower case bases are signal peptide nucleotide sequences, the underlined parts indicate the nucleotide sequences of CQ24-VHH, and the bolded parts indicate the nucleotide sequences of the human Fc fragment.

In the present invention, the backbone vector of the recombinant expression vector is preferably pcDNA3.1. The multiple cloning site for inserting the coding gene into the backbone vector is preferably Not1/Xba1.

The construction method of the recombinant expression vector is not particularly limited, and the construction method of constructing the recombinant expression vector well known in the art may be adopted.

In the present invention, the host bacteria of the engineering bacteria preferably comprise a prokaryotic expression system and/or a eukaryotic expression system. The prokaryotic expression system preferably comprises an E.coli strain. The eukaryotic expression system preferably comprises a yeast strain.

The construction method of the engineering bacteria is not particularly limited, and is finished by adopting construction methods of engineering bacteria known in the art, such as a chemical reagent conversion method and an electric shock conversion method.

The invention obtains the nano antibody fusion protein through recombinant expression of the engineering bacteria, has higher expression quantity, and has extremely high affinity when ELISA detects the binding of the nano antibody fusion protein and the prolactin receptor, and has EC ₅₀ 4.6ng/mL.

Based on the high affinity of the nanobody and the prolactin receptor, the invention provides application of the nanobody, the nanobody fusion protein, the coding gene, the recombinant expression vector or the engineering bacteria in preparing medicaments for inhibiting the binding of the prolactin and the prolactin receptor.

In view of the fact that the nanobody can effectively inhibit the combination of the prolactin and the prolactin receptor, and meanwhile, diseases are often caused by abnormal content of the prolactin and the prolactin receptor, the invention provides application of the nanobody, the nanobody fusion protein, the coding gene, the recombinant expression vector or the engineering bacteria in preparing medicines for preventing and/or treating diseases caused by abnormal content of the prolactin or the prolactin receptor.

In the invention, the nanobody reduces the activation of downstream signal channels of the prolactin receptor by competing with the prolactin to bind to the prolactin receptor, thereby inhibiting diseases caused by abnormal prolactin receptor. The disease preferably comprises at least one of the following: infertility, galactorrhea, prolactinoma, amenorrhea, menoxenia, vision disorders, hypopituitarism, systemic lupus erythematosus, systemic scleroderma, behcet's disease, drying syndrome, psoriasis, hypercarrheaemia, acne and breast cancer.

Clinical studies have demonstrated that dysgalactiae lead to infertility, galactorrhea, prolactinoma, amenorrhea, menoxenia, vision disorders, hypopituitarism, systemic lupus erythematosus, systemic scleroderma, behcet's disease, sjogren's syndrome, psoriasis, hyperchlorhydria, acne and breast cancer.

The dosage form and the preparation method of the drug are not particularly limited, and the dosage form and the preparation method of the drug related to the antibody, which are well known in the art, can be adopted.

The present invention provides a nanobody against a prolactin receptor, a recombinant vector, a recombinant bacterium and applications thereof, which are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Construction method and screening method of phage display library

1. Recombinant expression preparation method of prolactin receptor antigen

The human prolactin receptor ECD fragment of 25-234aa is selected as the prolactin receptor antigen, the amino acid sequence is shown as SEQ ID NO. 6, the nucleotide sequence is shown as SEQ ID NO. 7, and the sequence is synthesized by Kangzhou sequencing part. Cloning the coding gene of the prolactin receptor antigen to a Not1/Xba1 polyclonal site of a mammalian cell expression vector pcDNA3.1, carrying out sequencing identification, and transferring the obtained recombinant vector into HEK293F cells for expression, and separating and purifying recombinant proteins to obtain the prolactin receptor antigen.

The amino acid sequence shown in SEQ ID NO. 6 is as follows:

MHSSALLCCLVLLTGVRAQLPPGKPEIFKCRSPNKETFTCWWRPGTD

GGLPTNYSLTYHREGETLMHECPDYITGGPNSCHFGKQYTSMWRTYIMMVNATNQMGSSFSDELYVDVTYIVQPDPPLELAVEVKQPEDRKPYLWIKWSPPTLIDLKTGWFTLLYEIRLKPEKAAEWEIHFAGQQTEFKILSLHPGQKYLVQVRCKPDHGYWSAWSPATFIQIPSDFTMNDIEGRMDHHHHHH。

the nucleotide sequence shown in SEQ ID NO. 7 is as follows:

wherein, the italic sequence is signal peptide, the underlined human prolactin receptor ECD (PRLR-ECD), the bold sequence is the FactorXa site, and the dotted line sequence is the 6 XHis tag sequence.

2. Alpaca immunity

Mixing the obtained prolactin receptor antigen with Freund's adjuvant according to the volume ratio of 1:1, and emulsifying to obtain the vaccine.

The prepared vaccine alpaca neck vein is injected at 2 points, and each point is injected with 0.4mL of mixed solution. The immunization was repeated every 2 weeks for a total of 4 times.

3. Construction of nanobody library from purified lymphocytes

After 4 immunizations of alpaca, 50mL were collected every seven days intravenously. 5mL of blood was drawn from each blood sampling for precooling, and then centrifuged at 25℃for 30 minutes, and the supernatant serum was removed for antibody effect evaluation. The separation solution was added to the centrifuge tube, then blood was added at a ratio of 1:1, followed by centrifugation for 30 minutes, and then the supernatant serum was stored in a fresh centrifuge tube, -80 degrees. The immune cells were removed from the tube, added with PBS buffer, centrifuged at 25℃for 20 minutes, the supernatant removed, and the lymphocyte solution was separated by lysis according to the number of cells, -80℃for storage. The lymphocytes extracted by us are dissolved and transferred to a new centrifuge tube, chloroform is added and mixed evenly, and the lymphocytes are transferred to the new centrifuge tube after centrifugation for 15 minutes. Adding isopropanol with the same volume, standing for 10 minutes, centrifuging again for 10 minutes, precipitating with 75% ethanol clearly, centrifuging for 5 minutes, removing supernatant, and adding the solvent without RN into the water of the enzyme A obtained above; and (3) transcription of cDNA.

RNA was split into two parts, transcribed into cDNA, and then amplified by PCR. Wherein the amplification primer:

F(CH2-R)：GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO:8)；

R(CA-LD)：CTTGGTGGTCCTGGCTGC(SEQ ID NO:9)；

the reaction system amounted to 50. Mu.l:

PCR reaction conditions: pre-denaturation at 98 ℃ for 1min; 15s at 98 ℃, 20s at 56 ℃ and 1min25s at 72 ℃;30 cycles; preserving heat at 4 ℃.

The PCR amplification product obtained was cloned into phage vector pComb3xss at the Sfi site: the PCR amplification product and phage vector were digested, purified and subjected to ligation reaction. And purifying and recovering the enzyme digestion products respectively, dissolving the enzyme digestion products by using ultrapure water, and connecting the enzyme digestion products under the action of ligase. The ligation products and helper phage were mixed in a ratio of 20:1 by number and incubated with the cells for 30 minutes, kanamycin at a concentration of 50ug/ml was added and shake incubated at 30 ℃. The resulting cultured cells were centrifuged for 5min, and the supernatant was separated, and after 30min ice compress, centrifuged for 10min and the dissolved precipitate in the buffer was removed. Adding NaCl solution, incubating on ice for 10 minutes, and centrifuging for 15 minutes to finally obtain a phage library;

4. phage affinity panning

The antigen protein was diluted to 5. Mu.g/ml with PBS, 100. Mu.l/well was added to the microplate wells and coated at 4℃for 12h. The coating was discarded, washed 3 times with PBS, and each well was blocked with 300. Mu.l of 3% BSA-PBS blocking solution at 37℃for 2h. The PBS was washed 6 times.

100 μl phage library was added to the blocked ELISA plate, and the number of phages was about 2×10 ¹¹ CFU,37 ℃ incubation 2h. Unbound phage were aspirated, washed 5-15 times with PBST (2 min each rest), and 2-5 times with PBS.

To the washed ELISA plate, 100. Mu.l Gly-HCl and 1mg/ml BSA (pH=2.2) were added and incubated at 37℃for 12min.

Specifically bound phage were eluted, the eluate was transferred to a sterile centrifuge tube, rapidly neutralized with 10% by volume Tris-HCl (ph=9.1) as an eluent and the eluted product was stored at 4 ℃.

Mu.l of the library after panning was taken and plated on E.coli culture. TG1 to OD was cultured in2 XYT medium to 0.4 to 0.8. Ext> mixingext> elutriationext> eluateext> withext> 18ext> mlext> TGext> inext> theext> earlyext> stageext> ofext> logarithmicext> growthext>,ext> shakeext> culturingext> atext> 37ext> deg.Cext> forext> 45ext> minext> atext> 220ext> rext> /ext> minext>,ext> transferringext> toext> 15ext> mlext> 2ext> XYText> -ext> Gext> -ext> Aext> liquidext> cultureext> mediumext>,ext> culturingext> atext> 37ext> deg.Cext> forext> 2ext> hrext> atext> 220ext> rpmext>,ext> addingext> 100ext> μlext> ofext> auxiliaryext> phageext>,ext> standingext> atext> 37ext> deg.Cext> forext> 15ext> minext>,ext> andext> shakeext> culturingext> atext> 220ext> rpmext> forext> 30ext> -ext> 45ext> minext>.ext> The cultures were dispensed into centrifuge tubes and centrifuged at 3500rpm at 4℃for 10min, and the cell pellet was resuspended in25 ml 2 XYT-AK liquid medium and shake cultured at 250rpm at 30℃overnight. The overnight culture was centrifuged at 12000rpm for 20min at 4℃and the supernatant was transferred to a new centrifuge tube, 5-half volume of PEG-NaCl was added, and the mixture was stirred and placed at 4℃for 1h or more. The supernatant was removed at 12000rpm for 20min at 4℃and the pellet was resuspended in 300-500. Mu.l PBS and titered was determined for the next round of panning or analysis. From the final round of elutriation eluate titer plates (number not 30-200), several single colonies were randomly picked with sterilized toothpicks in 170. Mu.l 2 XYT-amp medium (placed in 2ml 96 Kong Miejun deep well plates, sealed with plate films to prevent mutual contamination) and shake-cultured at 37℃for 3h at 220 rpm. 100ul of helper phage was added to 100ml of 2 XYT-amp, and after mixing, 170. Mu.l of the mixture was added to each well Shan Junla, and after standing at 37℃for 15min, shaking culture was performed at 220rpm for 30 to 45min. To the medium was added 170. Mu.l of 2 XYT-amp and 3 times of 50mg/ml kanamycin, and the final concentration of kanamycin in 510. Mu.l of the medium was 50. Mu.g/ml, and the medium was incubated at 30℃and 220rpm overnight. Target protein was diluted to 2. Mu.g/ml with PBS, added to the ELISA plate at 100. Mu.l/well, and coated overnight at 4 ℃. The coating solution was discarded, PBST (tween-20 concentration 0.1%, the same applies hereinafter) was washed 3 times, 300. Mu.l of 3% skim milk was added to each well, and the wells were blocked at 37℃for 2 hours. The blocking solution was discarded, washed 5 times with PBST, and 50. Mu.l of the supernatant of the monoclonal bacteria solution (the bacteria solution was centrifuged at 3800rpm at 4℃for 20 min) was added and then the mixture was blown off several times and incubated at 37℃for 1 to 2 hours. The waste liquid is discarded, washed 5 times by PBST (about 2min in each middle standing), secondary antibody (diluted by 3% skimmed milk) is added, 100 μl/hole is added, and incubation is carried out at 37 ℃ for 30 min-1 h. The waste liquid is discarded, PBST is washed for 5 times, a reaction substrate TMB chromogenic solution is added for color development, 50 ul/hole is added, color development is carried out at 37 ℃ for 5 min-15 min, 50ul of stop solution is added for stopping reaction, and the value is read by an enzyme label instrument.

TABLE 1 sample numbers 1-92 positions

1	9	17	25	33	41	49	57	65	73	81	89
												2	10	18	26	34	42	50	58	66	74	82	90
3	11	19	27	35	43	51	59	67	75	83	91
												4	12	20	28	36	44	52	60	68	76	84	92
5	13	21	29	37	45	53	61	69	77	85	Control
												6	14	22	30	38	46	54	62	70	78	86	Control
7	15	23	31	39	47	55	63	71	79	87	Control
												8	16	24	32	40	48	56	64	72	80	88	Control

TABLE 2 sample Nos. 1-92 detection results

0.052	0.199	3.114	0.561	3.198	0.425	0.117	0.213	0.162	0.23	0.268	0.17
												0.171	0.158	3.272	0.506	3.216	0.568	0.046	0.052	0.048	0.053	0.272	0.273
0.251	0.164	0.076	0.636	3.251	0.294	0.188	0.244	0.238	0.142	0.041	0.51
												0.059	0.181	0.041	0.499	2.197	0.162	0.226	0.231	0.233	2.449	0.144	0.494
0.281	1.124	0.162	0.107	0.176	0.046	0.052	0.048	0.053	0.047	0.35	0.009
												0.093	2.951	0.072	0.217	0.057	2.334	2.385	0.387	3.415	0.177	3.373	0.067
0.183	1.593	3.344	0.212	0.058	1.474	0.521	2.501	0.128	0.197	0.08	0.027
												0.193	0.041	2.926	0.043	0.044	0.065	0.291	0.284	3.406	3.393	0.135	0.028

ELISA detection of monoclonal Positive Rate after affinity panning:

1) 4 human prolactin receptor-ECD (2. Mu.g/ml) protein plates coated overnight at 4℃were blocked with 5% nonfat dry milk and incubated for 2h at 37 ℃; washing with PBS for 2 times after the end of the sealing;

2) The bacteria liquid cultured by CQ-ECD deep hole plate is centrifuged for 10min at 3800rpm, so that the bacteria are separated from the culture supernatant (taken as a sample);

3) Taking newly coated FC-S2 protein plates, adding 50 μl of 5% skimmed milk powder respectively, then sucking 50 μl of sample, mixing with skimmed milk powder at equal ratio, and incubating at 37deg.C for 1 hr;

4) Washing with PBST for 5 times, and drying the 96-well plate in a beating way;

5) The dose range recommended by the anti-M13 anti-body (HPR) Antibody specification was 0.1-0.4 μg/ml, diluted Antibody (1: 7500 dilution) was used as secondary antibody, 100 μl of secondary antibody was added to each well, and incubated at 37deg.C for 45min;

6) Washing with PBST for 5 times, and drying the 96-well plate in a beating way;

7) Adding 100 μl of color development liquid into each hole, standing for 5min, and stopping with stop liquid when the color difference between the negative control hole and the positive hole is obvious;

8) Reading on the enzyme label instrument at 450nm;

single colonies with positive detection values (OD value is more than 0.2) and higher read value sequences are randomly picked from each deep hole plate for sequencing, and the sequencing primers are lacF: ACACTTTATGCTTCCGGCTCG (SEQ ID NO: 10) to give the sequence CQ01-CQ30, wherein the amino acids are shown in Table 1.

TABLE 1 amino acid sequence information of CQ01-CQ30 nanobody

Example 2

CQ24 nanobody expression and identification

The experimental steps are as follows: the protocol was used according to the instructions for the use of the KOP293 transient transfection protein expression system (version 3.3) from the pearl sea .

1. Construction method of recombinant vector

The nanobody obtained as described above was fused with a signal peptide, a connecting peptide and an Fc region to form a fusion protein of a signal peptide-connecting peptide-nanobody-Fc region. The fusion gene fragment corresponding to the fusion protein is cloned to the Not1/Xba1 multiple cloning site of the expression vector pcDNA3.1. And (3) transforming the recombinant expression vector by using escherichia coli, screening positive clones, and extracting plasmids to obtain the recombinant expression vector.

2. Cell culture prior to transfection: HEK-293 cells were placed in 5% CO ₂ In a constant temperature shaker (using other concentrations of CO) ₂ Can seriously affect the cell culture effect), and shake-culturing at a constant temperature of 120rpm at 37 ℃. In the process of passage, the strain is prepared,firstly, cell counting and observing cell activity rate are needed, and the density is selected to be (3-6) multiplied by 10 as much as possible ⁶ High-viability cells per ml were subcultured. Cells with excessively high culture density may have slow growth rate, reduced culture density and other growth state changes after subculture, and may directly affect the subsequent application effects of recombinant protein expression and the like.

3. Transient transfection (taking transfection of 100ml cell suspension as an example)

a. Preparing two 15ml sterile centrifuge tubes, adding 5ml KPM and 100 μg sterile recombinant vector into one of the two sterile centrifuge tubes, and gently blowing and mixing; taking the other separation tube, adding 5ml KPM and 500 μl TA-293 transfection reagent, gently blowing and mixing;

b. transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and lightly blowing and uniformly mixing;

c. standing for 10 minutes at room temperature to prepare a plasmid-carrier compound;

d. taking out cells from the constant temperature shaking table, adding the prepared plasmid-carrier complex while shaking, and returning to CO ₂ Shake culturing in a constant temperature shaking table. After 3 hours, an appropriate amount of antibiotics can be added as required. The expression level of the product was measured on the 6 th day after transfection.

The results are shown in Table 2.

TABLE 2 expression results of different nanobody fusion proteins

Example 3

Purification of CQ24 nanobodies

Cell supernatants were collected about 6 days after plasmid transfection into 50mL centrifuge tubes, centrifuged at 8000rpm for 15min, and the supernatants were collected. 1mL of a packing was charged into an empty chromatography column (column volume: 6 mL), and a balance solution, an eluent, a neutralization solution, and the like were prepared. Purification was performed according to the procedure described in table 3. It is recommended to store 0.5-1.0mL of sample per step for purity and concentration detection.

Table 3 purification procedure

Reagent(s)	Volume of
		Washing with water	10mL
0.1MNaOH	3mL
		Washing with water	10mL
Balancing liquid	5mL
		Loading sample	30mL
Balancing liquid	10mL
		Eluent (eluent)	1/10 of the neutralization solution is added into the collection bottle and 5mL of the neutralization solution is added
0.1MNaOH	3mL
		Washing with water	10mL
Balancing liquid	5mL
		Washing with water	10mL
20% ethanol	5mL

And concentrating the collected eluent by using ultrafiltration tubes such as 30KD or 50KD according to the molecular weight of the target protein, and replacing a buffer system of the target protein by PBS for at least 100 times. And (3) detecting the purity and the concentration of the finally obtained nano antibody, subpackaging, and preserving at-20 ℃.

The results are shown in Table 4.

TABLE 4 purity and concentration detection results

Example 4

ELISA high affinity assay of nanobody affinity

The indirect enzyme-linked immunosorbent assay is used for determining the binding capacity of CQ24 nano-antibodies to antigens (prolactin receptor), and comprises the following specific methods:

antigen (prolactin receptor) protein was diluted to 0.1. Mu.g/mL with coating solution (phosphate coating buffer, pH 7.4), 100. Mu.L/well was added to the ELISA plate and coated overnight at 4 ℃. TBST plates were washed 3 times, 200. Mu.L/well blocking solution (1.5% BSA) was added, and after incubation at 37℃for 2h TBST plates were washed 3 times. The purified nanobody was diluted to 1. Mu.g/mL with 1.5% BSA, and after 1:3 gradient dilution, the ELISA plate was added, 50. Mu.L/well, and incubated at 37℃for 1h. Washing the plate for 3 times by TBST, adding HRP-marked goat anti-mouse secondary antibody, and incubating for 60min at 37 ℃; after washing the plate 3 times with TBST, the residual liquid drops are beaten as much as possible on the absorbent paper, 50 μl of TMB is added into each hole, and the plate is placed in a dark place at 37 ℃ for 5min; add 25. Mu.L 2M H to each well ₂ SO ₄ Stop solution stops substrate reaction, enzymeOD values were read at 450nm in a standard meter and CQ24 nanobodies were analyzed for their ability to bind to antigen (prolactin receptor). Recording the OD value, drawing a fitting curve by combining the antibody concentration, and finally finding a point of half of the strongest signal, wherein the corresponding antibody concentration is EC ₅₀ . Wherein EC is ₅₀ Defined as the concentration that produces 50% efficacy or binding.

The results are shown in Table 5.

TABLE 5 affinity assay results

The results show that CQ24 nanobody binds human prolactin receptor-ECD protein with high affinity, EC ₅₀ 4.6ng/mL (see FIG. 1), superior to other nanobodies.

Example 5

Determination of blocking the prolactin receptor signalling pathway (different protein treatment concentrations)

Western immunoblotting assay nanobodies (CQ 24, CQ25, CQ27, CQ 28) block the downstream signaling pathway of human prolactin receptor as follows:

T47D cells were starved for 2 hours. The old medium was pipetted off, 0.5ml of sterile PBS solution was added, the mixture was swirled and spun down, PBS was pipetted off, and the mixture was washed 3 times. The serum was thoroughly washed off, and 0.5ml of serum-free cell culture medium was added and placed in an incubator. The operation was performed for about 20 minutes. Starvation culture was carried out at 37℃for 2 hours. The desired protein treatment concentration is calculated, and the added volume of purified protein is calculated according to the treatment concentration and the purified protein concentration. The operation was performed for about half an hour. Purified protein was added to supplement BSA, PRL, as calculated by treatment concentration. Mixing the materials upside down, filtering and sterilizing. After 2 hours of constant temperature treatment in a 37 ℃ incubator, the cells are collected. 100ul RAPI is cracked for 5 minutes, and a cracking solution is collected. Repeatedly shaking, centrifuging, and blowing broken DNA for about 10 times. Adding 5×loading buffer, mixing, and centrifuging. After thermal denaturation of the protein samples for 10min, SDS-PAGE electrophoresis was performed. Semi-dry transfer for 10 minutes. 5% nonfat dry milk was blocked for 1 hour. STAT5-P primary antibody was incubated at 4 ℃ overnight. TBST was washed three times. The secondary antibody was incubated for one hour at room temperature. After three times of TBST washing, chemiluminescent development was performed.

The results are shown in FIG. 2. With increasing CQ24 nanobody treatment concentration, phosphorylation of downstream signaling pathway STAT5 was inhibited.

The results of fig. 3 show that only CQ24 nanobodies have the effect of inhibiting phosphorylation of downstream signaling pathway STAT5 compared to other nanobodies.

Comparative example 1

ELISA determination of affinity of conventional antibodies

The indirect enzyme-linked immunosorbent assay measures the binding capacity of a conventional antibody to an antigen (prolactin receptor) as follows:

antigen (prolactin receptor) protein was diluted to 0.1. Mu.g/mL with coating solution (phosphate coating buffer, pH 7.4), 100. Mu.L/well was added to the ELISA plate and coated overnight at 4 ℃. TBST plates were washed 3 times, 200. Mu.L/well blocking solution (1.5% BSA) was added, and after incubation at 37℃for 2h TBST plates were washed 3 times. A conventional antibody (anti 005-C04 in which the amino acid sequence of VH is SEQ ID NO:40 (EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLE WVSDISSASSYTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CARGLDARRMDYWGQGTLVTVSS) and the amino acid sequence of VL is SEQ ID NO:41,QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYVVHWYQQLPGTAPKLLIY RNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGW LFGGGTKLTVLGQ) was diluted to 1. Mu.g/mL with 1.5% BSA, and after 1:3 gradient dilution, an ELISA plate was added, 50. Mu.L/well and incubated at 37℃for 1h. Washing the plate for 3 times by TBST, adding HRP-marked goat anti-mouse secondary antibody, and incubating for 60min at 37 ℃; after washing the plate 3 times with TBST, the residual liquid drops are beaten as much as possible on the absorbent paper, 50 μl of TMB is added into each hole, and the plate is placed in a dark place at 37 ℃ for 5min; add 25. Mu.L 2M H to each well ₂ SO ₄ The stop solution stops the substrate reaction, the OD value is read at 450nm of the enzyme label instrument, and the binding capacity of the conventional antibody and antigen (prolactin receptor) is analyzed. Recording the OD value, drawing a fitting curve by combining the antibody concentration, and finally finding a point of half of the strongest signal, wherein the corresponding antibody concentration is EC ₅₀ . Wherein EC is ₅₀ Defined as the concentration that produces 50% efficacy or binding.

The results are shown in FIG. 4. Nanobodies have a higher affinity than conventional antibodies.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The nanometer antibody of the anti-prolactin receptor is characterized in that the amino acid sequence of the nanometer antibody is shown as SEQ ID NO. 1.

2. The nano antibody fusion protein of the anti-prolactin receptor is characterized in that the nano antibody fusion protein is a signal peptide-connecting peptide-the nano antibody-Fc region of claim 1, and the amino acid sequence is shown as SEQ ID NO. 2.

3. A coding gene of the nanobody fusion protein as claimed in claim 2, wherein the nucleotide sequence of the coding gene is shown in SEQ ID NO. 3.

4. A recombinant expression vector comprising the coding gene of claim 3.

5. The recombinant expression vector according to claim 4, wherein the backbone vector of the recombinant expression vector is pcDNA3.1.

6. The recombinant expression vector of claim 5, wherein the multiple cloning site for insertion of the coding gene into the backbone vector isNot1/Xba1。

7. An engineered bacterium comprising the recombinant expression vector of any one of claims 4 to 6.

8. The engineering bacterium according to claim 7, wherein the host bacterium of the engineering bacterium comprises a prokaryotic expression system and/or a eukaryotic expression system.

9. Use of a nanobody according to claim 1, a nanobody fusion protein according to claim 2, a coding gene according to claim 3, a recombinant expression vector according to any one of claims 4 to 6 or an engineering bacterium according to claim 7 or 8 for the preparation of a reagent for inhibiting the binding of prolactin to a prolactin receptor.

10. Use of the nanobody of claim 1, the nanobody fusion protein of claim 2, the encoding gene of claim 3, the recombinant expression vector of any one of claims 4 to 6, or the engineering bacterium of claim 7 or 8 in the preparation of a medicament for preventing and/or treating a disease caused by an abnormality of a prolactin or a prolactin receptor;

the disease includes at least one of: infertility, galactorrhea, prolactinoma, amenorrhea, menoxenia, vision disorders, hypopituitarism, systemic lupus erythematosus, systemic scleroderma and hyperlactation.