CN117924482A - Nano antibody targeting INH alpha and application thereof - Google Patents

Abstract

The invention provides a nanobody targeting INH alpha and application thereof, and belongs to the technical field of nanobodies. According to the invention, 4 anti-INH alpha nanobody genes are obtained from camel serum through screening by phage display technology for the first time, and the purified anti-INH alpha nanobody proteins are obtained through construction of prokaryotic expression vectors, protein induced expression, purification and identification. The nano antibody provided by the invention can be used for preparing the sheep inhibin immunosuppressant, and solves the problem that the current inhibin immunosuppressant cannot be popularized and used due to the defects of low immunogenicity, poor stability, poor solubility, difficult expression and the like.

Description

Nano antibody targeting INH alpha and application thereof

Technical Field

The invention belongs to the technical field of nanobodies, and particularly relates to a nanobody targeting INH alpha and application thereof.

Background

Xinjiang is one of four pastures nationwide, not only has excellent grasslands with the largest nationwide area, but also has a plurality of excellent local sheep varieties, is the largest sheep variety gene library in China, and most of the local sheep varieties have single production performance. Xinjiang has a lack of excellent local sheep varieties which can adapt to local environment and can be produced comprehensively. Kazakh sheep is used as a local sheep variety resource in Xinjiang, and has strong environmental adaptability and strong cold resistance and disease resistance. The sheep has the advantages of early maturing, high and cold resistance, coarse feeding resistance, quick growth and development, high survival rate of the lambs and the like, is deeply favored by peasants and herders in the local area, and is an excellent local sheep variety for meat. However, kazakh sheep like most of meat sheep breeds in Xinjiang, although a part of Kazakh sheep can heat in spring and autumn under good feeding management conditions, the total breeding rate is far lower than that of sheep breeds in other excellent places such as Hu sheep and Qira black sheep. This has severely affected the enthusiasm of farmers to breed Kazakhstan sheep to some extent. Hybridization with other sheep varieties with high reproductive rate can improve the lambing rate of Kazakh sheep to a certain extent, but the meat yield, stress resistance and environmental adaptability of Kazakh sheep are obviously reduced after hybridization. Therefore, a method for effectively improving the reproductive performance of the sheep is searched, and the method has important significance for protecting, developing and utilizing the Xinjiang excellent meat local sheep variety.

Lambing rate is always a major factor affecting sheep reproductive performance, and the number of lambing in sheep depends on the number of eggs discharged per estrus of sheep, which means that the key to increase reproductive rate is how to increase ovulation number in sheep in estrus. Previous studies have shown that the number and magnitude of ovulation in sheep is positively correlated with the amount of Follicle Stimulating Hormone (FSH) expressed in the body during estrus. Whereas single-born animals, like multiple-born animals, have the physiological basis of superovulation, except that long-term natural selection allows them to secrete only FSH and Luteinizing Hormone (LH) required for the development and maturation of individual follicles during oestrus. Thus, if the amount of endogenous FSH secreted during estrus in sheep can be increased by a certain method, it is possible to significantly increase the number of ovulations during estrus in sheep, i.e. a corresponding increase in the number of lambings in sheep. This requires starting from the intrinsic regulatory mechanisms of FSH secretion. Currently, inhibin (Inhibin, INH) is considered to be a major factor in regulating FSH secretion in vivo. The hormone is secreted mainly by granulosa cells of the ovary in females and the main physiological effect is negative feedback inhibition of pituitary FSH secretion, which has been studied to show that inhibin plays an important role in follicular growth, development, occlusion and ovulation processes, but its specific regulatory mechanism is not yet known.

Inhibin was originally isolated from bovine follicular fluid as a macromolecular glycoprotein hormone, a heterodimer formed by two different subunits, the alpha and beta subunits, linked by disulfide bonds, and inhibin A (INHA) and inhibin B (INHB) with biological activity, respectively. It is an important hormone of the hypothalamic-pituitary-gonadal axis regulation system, which has a very close relationship with reproductive endocrine, and was originally named for the inhibition of pituitary follicle stimulating hormone production and secretion by autocrine and paracrine forms. The existing research shows that Inhibin alpha subunit gene (Inhibin alpha, INH alpha) is a main effective gene for negatively regulating the growth and development of animal follicles, and the gene function may have conservation among different species. This provides the possibility of breeding livestock molecules by mutation using INH alpha as a target gene or improving sheep breeding efficiency through active and passive immunization routes of gene expression products. At present, the study on improving the fertility of cattle and sheep by using inhibin immunization is more. It was concluded that essentially the immunization with inhibin partially increased the fertility of the animals. However, the technology is difficult to be widely applied in production due to objective reasons such as difficult preparation of pure inhibin protein or preparation of inhibin monoclonal antibody, complicated preparation process, high cost, inconvenient use and the like.

The birth and development of the gene vaccine provides a new opportunity for inhibin immunization. Genetic immunization is also known as DNA immunization or nucleic acid immunization. The inhibin DNA immunity is based on gene immunity and inhibin conventional immunity, and the exogenous gene for coding inhibin antigen determinant is converted into animal body by means of recombinant expression vector, so that the target gene can be used for synthesizing antigen protein by means of transcription system of host cell, inducing host to produce antibody and inducing correspondent specific immune response, and can reduce in vivo inhibin level to a certain extent so as to improve ovulation and sperm effect of animal. However, the main problems of eukaryotic recombinant proteins and prokaryotic expression products are low immunogenicity of the recombinant proteins, unsatisfactory immune effects and the like, which affect the practical application of the recombinant proteins. In addition, the INH genes are constructed on plasmids containing resistance marks, so that certain potential safety hazards exist, and the INH genes cannot be popularized and applied in large scale in actual production. And by utilizing a prokaryotic expression system, the target protein is obtained through the induction and purification of an in vitro prokaryotic expression vector, and the interspecies animals are immunized, so that the immune serum with high titer, namely the so-called polyclonal antibody, is finally obtained. Because the antibody components in serum are complex and various, the immune effect is often unstable. The inhibin traditional monoclonal antibody has complex preparation process and high cost, and can not be popularized in large scale in production. Thus, the search for new methods and new immunization strategies for preparing inhibin immune formulations has become a research hotspot for inhibin immune propagation technology. The technical scheme for solving the problem that the current inhibin immune preparation cannot be popularized and used due to the defects of low immunogenicity, weak stability, poor solubility, difficult expression and the like is needed to be provided in the field, and a foundation is laid for finally obtaining the economic and efficient sheep inhibin immune inhibitor.

Disclosure of Invention

Therefore, the invention aims to provide the INH alpha-targeted nano antibody which can be used for preparing inhibin immunosuppressants and solves the problem that the current inhibin immunosuppressants cannot be popularized and used due to the defects of low immunogenicity, poor stability, poor solubility, difficult expression and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a nanobody targeting INH alpha, which is at least one of Nb-4, nb-15, nb-26 and Nb-57; the amino acid sequences of Nb-4, nb-15, nb-26 and Nb-57 are respectively shown as SEQ ID NO.1-SEQ ID NO. 4.

The invention also provides a gene for encoding the nano antibody, and the nucleotide sequences of the genes for encoding the Nb-4, nb-15, nb-26 and Nb-57 nano antibodies are respectively shown as SEQ ID NO.5-SEQ ID NO. 8.

The invention also provides an expression vector capable of expressing the genes.

Preferably, the expression vector is pET-32a.

The invention also provides a host cell containing the expression vector.

Preferably, the host cell is E.coli BL21 (DE 3).

The invention also provides an application of the nano antibody or the gene or the expression vector or the host cell in preparing an anti-inhibin immune preparation.

The invention also provides an application of the nano antibody or the gene or the expression vector or the host cell in improving sheep lambing rate.

The invention has the beneficial effects that:

According to the invention, 4 anti-INH alpha nanobody genes are obtained from camel serum through screening by phage display technology for the first time, and the purified anti-INH alpha nanobody proteins are obtained through construction of prokaryotic expression vectors, protein induced expression, purification and identification. The nano antibody provided by the invention can be used for targeting and combining INHalpha, has high affinity, can inhibit the expression of inhibin, can be used for carrying out passive immunization on sheep, and can obviously reduce the level of INHA and INHB and obviously improve the level of FSH, so that the nano antibody disclosed by the invention can be used for preparing the sheep inhibin immunosuppressant, and is beneficial to solving the problem that the current inhibin immunosuppressant cannot be popularized and used due to the defects of low immunogenicity, poor stability, poor solubility, difficult expression and the like.

Compared with the traditional antibody, the nano antibody has the advantages of small molecular weight, good degradation resistance and stability, availability of prokaryotic expression, low mass production cost, easy product characterization, low post-translational modification, good uniformity, good tissue penetrability and easy absorption.

Drawings

Fig. 1: target gene amplification results, M in the figure is DL2000 DNA MARKER;1 is a negative control (ddH ₂ O as PCR template); 2. 3 is INH alpha PCR amplification product;

fig. 2: bacterial liquid PCR identification results, wherein M is DL2000 DNA MARKER; 1. 2,3 and 4 are INH alpha-pet 32a bacterial liquid PCR products; 5 is a negative control (ddH ₂ O as PCR template);

fig. 3: the prokaryotic expression vector enzyme digestion identification result, wherein M is standard DNAMARKER; 1.2 and 3 are double enzyme cutting electrophoresis results;

Fig. 4: SDS-PAGA electrophoresis analysis result of recombinant protein, wherein M is protein Marker; 1-5, 0.1mmol/L IPTG induction expression 0, 2,4, 6 and 8h protein electrophoresis results are sequentially carried out;

Fig. 5: a target protein Western-blot detection result, wherein M is a protein Marker;1 is BL21 with empty vector, 2-5 are INH alpha recombinant protein;

Fig. 6: first round PCR results of natural library, wherein M is DL2000 DNA MARKER; 1-6 are all first round nested PCR amplification products, and 1-6 are parallel test results;

fig. 7: the second round of PCR results of the natural library, wherein M is DL2000 DN AMARKER; 1-5 are second round nest PCR amplified products, 6 are blank controls;

fig. 8: ELISA titer detection of INH alpha specific antibodies;

Fig. 9: the library nest PCR result after immunization is a first round PCR result and a second round PCR result from left to right, M is DL2000 DNA MARKER in the left graph, 1-3 are first round nest PCR amplification products, and 4 is blank control; in the right graph, M is DL2000 DNA MARKER, and 1 to 6 are all second round nested PCR amplification products;

Fig. 10: bacterial liquid PCR identifies positive transformants, the left graph shows the results of 12 clones, and the right graph shows the results of 12 clones;

Fig. 11: VHH sequence alignment;

Fig. 12: enrichment of specific phage in the ELISA detection and screening process of phage;

fig. 13: ELISA method for detecting the binding capacity of recombinant nanobody and INH alpha;

Fig. 14: alignment charts of amino acid sequences of nano antibodies;

fig. 15: interaction of INHα with Nb-4;

Fig. 16: a nanometer antibody gene PCR product electrophoresis chart, wherein M is BM5000 DNA MARKER; 1-4 are respectively Nb-4, nb-15, nb-26 and Nb-57 gene PCR amplified products;

fig. 17: electrophoretogram of pET-32a-Nb recombinant plasmid double enzyme digestion products, wherein M is BM5000 DNA MARKER; 1-4 are pCantab-Nb-4, 15, 26, 57 plasmid double enzyme cutting products respectively;

FIGS. 18 to 21 are respectively and sequentially Nb-4, nb-15, nb-26 and Nb-57 nanobody induction expression electrophoresis patterns, wherein M is a protein Marker, and lanes 1 to 5 are respectively protein electrophoresis results when the induction time of the corresponding nanobody is respectively 0h,2h,4h,6h and 8h; lanes 6-7 are the results of supernatant and precipitation electrophoresis after cell ultrasonication, respectively;

FIGS. 22-25 are ELISA affinity assay plots for Nb-4, nb-15, nb-26, and Nb-57 nanobodies, respectively, in sequence;

Fig. 26: graph of affinity results of 4 nanobodies with INH alpha at 0.122 μg/mL;

FIGS. 27-32 show the levels of INHA, INHB, FSH, LH, E and PROG hormones, respectively, in serum after immunization.

Detailed Description

The invention provides a nanobody targeting INH alpha, which is at least one of Nb-4, nb-15, nb-26 and Nb-57; the amino acid sequences of Nb-4, nb-15, nb-26 and Nb-57 are respectively shown as SEQ ID NO.1-SEQ ID NO. 4.

In the invention, the amino acid sequence of the nano antibody Nb-4 is shown as SEQ ID NO.1, the amino acid sequence of the nano antibody Nb-15 is shown as SEQ ID NO.2, the amino acid sequence of the nano antibody Nb-26 is shown as SEQ ID NO.3, and the amino acid sequence of the nano antibody Nb-57 is shown as SEQ ID NO. 4. The variable region (CDR) and constant region (FR) of the 4-strain nanobody of the invention are shown in FIG. 14. The 4-strain INH alpha-targeting nano antibodies provided by the invention have higher affinity with INH alpha protein, and can be specifically combined with different parts of INH alpha protein. The provided complementarity determining region 3 (CDR 3) amino acid sequence length of the nano antibody is about 22, which is favorable for the nano antibody to flexibly adjust the conformation thereof, improves the binding effect with antigen, has lower preparation cost and is favorable for wide application.

The nanobody is preferably prepared by the following method:

cDNA is reversely transcribed by extracting total peripheral blood lymphocyte RNA of Xinjiang Bactrian camel immunized for multiple times (inhibin prokaryotic expression products), nested PCR is carried out by two pairs of specific primers, and the antibody heavy chain variable region (VHH) sequence fragments in the blood of the camel are amplified and recovered. And then utilizing phage display technology to display the VHH sequence fragment of the camel at the position connected with the M13KO7 phage capsid protein III so as to prepare the camel VHH phage library. The phage library obtained was subjected to 3-5 rounds of panning by phage ELISA to screen phage clones that could bind specifically to INH alpha protein, and then sequenced to obtain the final target gene sequence. The phage clone strain is propagated to obtain crude INH alpha nano antibody extract, or the target gene sequence is subjected to prokaryotic expression, and the steps of purification, dialysis and the like are carried out to obtain the high-concentration INH alpha nano antibody.

The invention further detects the combination titer of the nano antibody and INH alpha, and the screening and the identification show that the nano antibody has the advantages of small molecular weight, strong stability, good solubility, easy expression and low immunogenicity, provides a new thought for further defining the molecular mechanism of inhibin in sheep reproduction regulation and control, and provides reference data for researching the improvement of the Xinjiang sheep reproduction efficiency by using an immune means.

The invention also provides a gene for encoding the nano antibody, and the nucleotide sequences of the genes for encoding the Nb-4, nb-15, nb-26 and Nb-57 nano antibodies are respectively shown as SEQ ID NO.5-SEQ ID NO. 8. The nucleotide sequence of the gene for encoding the Nb-4 nanobody is shown as SEQ ID NO.5, the nucleotide sequence of the gene for encoding the Nb-15 nanobody is shown as SEQ ID NO.6, the nucleotide sequence of the gene for encoding the Nb-26 nanobody is shown as SEQ ID NO.7, and the nucleotide sequence of the gene for encoding the Nb-57 nanobody is shown as SEQ ID NO. 8.

The invention also provides an expression vector capable of expressing the genes. In the present invention, the expression vector is preferably pET-32a. The specific source of pET-32a is not particularly limited, and products conventionally and commercially available in the art can be used.

The invention also provides a host cell containing the expression vector. In the present invention, the host cell is preferably E.coli BL21 (DE 3). The specific source of the escherichia coli BL21 (DE 3) is not particularly limited, and the product conventionally and commercially available in the field can be adopted.

The invention also provides an application of the nano antibody or the gene or the expression vector or the host cell in preparing an anti-inhibin immune preparation or improving the lambing rate of sheep.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In the following examples, conventional methods are used unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1

1. Cloning and sequencing identification of Kazakh sheep INH alpha gene:

Collecting Kazakh sheep testis tissue, extracting tissue total RNA, performing reverse transcription to form cDNA, and amplifying INH alpha gene by RT-PCR (primers are shown in Table 1, INH alpha-F and INH alpha-R), wherein the reaction procedure is as follows: 95 ℃ for 10min;95 ℃ for 40s;67 ℃,30s;72 ℃,80s;72℃for 10min. And (3) recovering the amplified product through gel cutting, connecting the amplified product with a T carrier, and carrying out bacterial liquid PCR (a reaction system is shown in Table 2) and sequencing identification.

TABLE 1INH alpha Gene primer sequences

TABLE 2INH alpha Gene PCR System

Transformants were identified by bacterial liquid PCR and positive clones were screened. The results showed that the amplified band was consistent with the target gene fragment size (FIGS. 1 and 2). And (3) selecting positive clones, sending the positive clones to sequencing identification, and comparing the sequencing result with GenBank NM_001308579.1 (sheep INH alpha gene sequence) to find that 2 bases are mutated, wherein the sequence homology is 99.98%, which indicates that the INH alpha gene cloning is successful.

Construction, identification and induced expression of INH alpha gene prokaryotic expression vector pET32a (+) -INH alpha

(1) Construction and identification of prokaryotic expression vector pET32a (+) -INHα

The INH.alpha.gene sequence was amplified using primers with cleavage sites (see INH.alpha. -EcoRI-F and INH.alpha. -HindIII-R of Table 1) and the laboratory-maintained bacteria transformed with pET-32a expression vector were cultured overnight. The next day was performed for pET-32a vector extraction. The two recombinant plasmids and the two expression vectors were then double digested with EcoR I and Hind III restriction enzymes. Then, the digestion result is detected by gel electrophoresis, and the objective digestion product is recovered. The recovered gene fragment was ligated into the PET expression system and placed in a thermostatically circulating water bath at 16 ℃ overnight. The pET32a (+) -INHα recombinant plasmid was transformed into competent cells DH 5. Alpha. For cloning by heat shock at 42 ℃. Finally, the recovered bacterial solution was spread on ampicillin solid plate medium and cultured overnight in an incubator at 37 ℃. Colonies were picked for PCR identification, and colonies positive for PCR identification were subjected to overnight amplification culture. The bacterial liquid plasmid was extracted the next day, and the plasmid was identified as correct by double enzyme digestion, and three parallel experiments were performed in total (see FIG. 3 for results). And then sent to sequencing and sequence alignment with the template.

(2) INH alpha protein induced expression and affinity detection

And (3) converting the INH alpha recombinant plasmid into E.coli BL21, and inducing by IPTG to express the INH alpha recombinant protein. The linked expression vector is transformed into escherichia coli DE3 for induced expression, and the existence form, the optimal expression time and the immunogenicity identification of the expressed protein are determined. A single band of the recombinant protein expressed by the whole sequence of the coding region of the INH alpha gene is detected by SDS electrophoresis identification (see figure 4). The Western-blot detection results showed (FIG. 5) that INH.alpha.protein was able to bind to His tag antibodies and that binding bands occurred at the same position and size as the INH.alpha.protein. This indicates that the prokaryotic expression vector was successfully constructed and normally expressed in prokaryotic cells.

3. Amplification and recovery of VHH gene fragments of natural library of unimmunized Bactrian camels

100Ml of anticoagulated whole blood was collected in the tail vein of Bactrian camel, lymphocytes were isolated, total RNA extracted and reverse transcribed to form cDNA prior to immunization against the first INH alpha protein. The cDNA was used as a template, the first amplification step was performed using a first pair of primers (primers shown in Call001 and Call002 of Table 3) (see FIG. 6), the fragments amplified in the previous step were used as templates, and the amplification step was performed using a second pair of primers (primers shown in VHH-Sfi I-F and VHH-Not I-R of Table 3) to obtain a target band of 450bp (see FIG. 7), and the target band was recovered as a Bactrian camel natural library gene fragment.

TABLE 3VHH Gene nested PCR primers

Large scale purification of INH alpha protein and immune Bactrian camel

The purified INH alpha recombinant protein was diluted to an effective concentration with physiological saline, and was continuously injected 6 times every 12 days, and when the antibody level reached a predetermined gradient, the immunization was stopped, and whole blood was collected (immunization program see Table 4). On day 7 after the last immunization, 200mL of bactrian camel whole blood was collected, part was used to collect lymphocytes, part was used to collect serum and serum antibody titers were detected by indirect ELISA. Results: the titer of inhα -specific antibodies in serum can reach 1:409600 (see FIG. 8).

TABLE 4INHα immunization program

Construction of INH alpha-VHH hybrid library

(1) Amplification of Bactrian camel INH alpha antigen-prone Gene library fragments

7 Days after the last immunization, 150mL of bactrian camel whole blood was collected and about 2X 10 ⁶ lymphocytes were extracted therefrom. Two rounds of nested PCR (primers see Table 3) gave a 450bp VHH fragment (see FIG. 9) and recovered, which was a bactrian camelINH alpha antigen-prone gene library fragment.

(2) Construction of helper phage hybrid VHH phage display vectors

The amplified natural library gene fragment of the Bactrian camel and the gene fragment of the Bactrian camel INH alpha inclined library are mixed, and then the phage antibody library is obtained through molecular biological steps such as electrophoresis, recovery, connection (phage display vector/pCantab E), electrotransformation and the like. After conventional library construction, a display library with a library capacity of 2.1X10 ⁸ CFU was obtained. The bacterial liquid PCR result shows that the positive cloning efficiency reaches 96% (23/24) (see FIG. 10). 5 were randomly picked for sequencing and no repeat was found using DNA MAN alignment (see fig. 11), indicating good library diversity.

The above results show that the antibody level of the invention has reached 1 by 6 immunization of the Bactrian camel: 409600 lymphocytes 2×10 ⁶ were extracted from whole blood. The antibody fragments with the size of 450bp are amplified by nested PCR, and the nanometer antibody library with the reservoir capacity of 2.1 multiplied by 10 ⁸ is obtained through connection and electrotransformation, and 96 percent of clones containing the antibody fragments are identified by bacterial liquid PCR. Conclusion: the invention successfully constructs the INH alpha nanobody mixed library and lays a foundation for later screening of INH alpha nanobodies.

Screening, preliminary identification and molecular simulation docking of INHα -specific nanobodies

(1) Screening and preliminary identification of INHα -specific nanobodies

And (3) fixing the INH alpha prokaryotic expression protein serving as an antigen on the bottom of an ELISA plate for screening the specific nano antibody. During screening, the titer of recombinant phage in each round of eluent is measured, so that the enrichment degree of each round can be obtained and the effect evaluation is carried out. The results are shown in Table 5, where the P/Input values increased as the screening intensity increased at the completion of the third round of screening, and the P/N values were very pronounced after the last round of screening.

Further detection of enrichment effect: the screening products obtained after each round of purification and elution are taken, and the ELISA detection is carried out by coating the ELISA plate with INH alpha recombinant protein, and the result shows that the OD _450nm value is also continuously increased along with the increase of the number of rounds (see figure 12), which shows that after 3 rounds of screening, the anti-INH alpha specific phage is obviously enriched. The medium after the third round of screening was taken, 96 colonies were picked, and the binding of 96 nanobody crude extracts to inhα was detected using ELISA, and the ELISA results showed that 90 out of 96 colonies were positive. The results in table 5 and figures 12 and 13 both demonstrate that inhα -specific nanobodies have been enriched.

TABLE 5 enrichment of specific phages during the screening procedure

The 6 strains with the highest (positive/negative) values in FIG. 13 were selected for sequencing analysis. As a result, 3 of the 6 clones were identical in sequence (pCantab E-Nb-4), and the other 3 clones were different from each other (pCantab E-Nb-15, pCantab 5E-Nb-26, pCantab 5E-Nb-57). The amino acid sequences of the 4 nano antibodies are aligned, and the result shows that the total similarity of the amino acid sequences of the 4 nano antibodies is 69% (see FIG. 14).

(2) Molecular docking simulation of recombinant proteins and antibody peptide fragments

Three rounds of panning were performed on inhα nanobodies by solid phase screening techniques. ELISA was performed to identify antibody affinities after three rounds of screening, 6 strains with highest affinities were selected, and the INH alpha and Nb-4 were subjected to simulated docking by HEX software, and the results show that the INH alpha protein has strong interaction with Nb-4 (FIG. 15), and the total free energy of the INH alpha protein is-576.28 kJ/mol.

The research results show that on the basis of prokaryotic expression of INH alpha recombinant protein and natural library construction of the Bactrian camel antibody, a 1.7X10 ¹¹ recombinant phage VHH library is finally obtained through rescue, culture and purification of the helper phage M13KO 7; after 3 rounds of panning were completed, phage ELISA results showed that phage enriched for 3 rounds increased significantly; the ELISA result of the nano antibody shows that the crude protein extracts of 96 positive clones can be combined with INH alpha to different degrees; 6 strains with highest affinity are selected for sequencing, and 4 INH alpha protein antibody peptide fragments are obtained; finally, the molecular simulation docking result shows that the elutriated nanobody Nb-4 has better binding capacity with INH alpha protein, and the binding free energy is-576.28 kJ/mol.

Example 2

Construction of nanobody gene prokaryotic expression vector

The screened pCantab E-Nb (Nb refers to Nb-4, nb-15, nb-26 and Nb-57) recombinant plasmid is taken as a template (the upstream primer and the downstream primer are respectively added with EcoR I and Hand III enzyme cutting sites, the upstream primer is 5'-CCGGAATTCCGGCAGGTCCAACTGCAGGAGTCT-3' (SEQ ID NO. 17) and the downstream primer is 5'-CCCAAGCTTGGGTGAGGAGACGGTGACCTGGGT-3' (SEQ ID NO. 18)) to carry out PCR amplification on target genes, and agarose gel electrophoresis and gel recovery are carried out on PCR products. The agarose gel electrophoresis result after PCR amplification shows that a specific target band of about 450bp is generated (figure 16), and the PCR product is consistent with the size of the target gene band.

And (3) connecting the Nb gene fragments subjected to double digestion with a pET-32a (+) vector, converting competent cells DE3, carrying out propagation on single bacterial colonies positive to bacterial liquid PCR, extracting plasmids, carrying out double digestion, displaying a 5900bp band and a 450bp band by agarose gel electrophoresis results (figure 17), and judging that 4 pET 32a-Nb recombinant plasmids are successfully constructed.

The results of SDS-PAGE electrophoresis after the induction of bacterial solutions at different times, the ultrasonic disruption of the supernatant and the mixture of the sediment and 5 XSDS-PAGE Loding Buffer (FIG. 18-FIG. 21) show that the protein expression level in the bacterial solutions is gradually increased along with the increase of time, which indicates that the induction and the expression of the recombinant protein of the 4-strain nanobody are successful. In addition, the amount of protein precipitated after ultrasonication was found to be higher than that of the supernatant, indicating that 4 nanobody proteins were mainly present in inclusion bodies.

Example 3

Nanobody and INH alpha protein affinity identification

Coating INH alpha protein as antigen 4 μg/Kong Baobei on 96-well ELISA plate (negative control antigen is PBS, positive control antigen is equally screened nanometer antibody protein) overnight, and sealing with 5% skimmed milk powder at 37deg.C for 2 hr; nanobody diluted with 2-fold gradient (250. Mu.g/mL, 125. Mu.g/mL, … …, 0.122. Mu.g/mL) was used as primary antibody and incubated at 37℃for 1h; taking an HRP-marked mouse anti-E-tag polyclonal antibody as a secondary antibody, and incubating for 1h at 37 ℃; after developing at room temperature for 15min using TMB single-component developing solution, a stop solution was added, and OD value was measured at a wavelength of 450 nm.

The ELISA affinity identification results are shown in figures 22-25, respectively, and the results show that 4 INH alpha specific nano antibodies all have higher affinity with INH alpha protein. Wherein Nb-4 was still able to bind INH alpha protein at 0.122. Mu.g/mL, which was the best affinity 1 strain of the 4-strain nanobody (FIG. 26).

Example 4

Sheep immunoassay

45 Adult sheep with 3-5 years of estrus time similar and in estrus are randomly divided into 4 groups, 15 adult sheep are respectively used as a inhibin alpha recombinant protein plus adjuvant immune group (A group), 15 inhibin alpha nano antibody Nb-4 recombinant protein immune group (B group), 15 normal saline group (C group), two immunizations are carried out on all sheep, 10mL of the jugular vein blood is collected before immunization, and inhibin alpha protein and Freund's adjuvant 1 are collected before immunization of the A group: 1 (Freund's complete adjuvant is used for the first immunization and Freund's incomplete adjuvant is used for the second immunization), and then subcutaneously injecting the mixture into sheep neck (1 mL/dose for the first immunization, 0.1mg/mL protein concentration, 0.5 mL/dose for the second immunization, 0.1mg/mL protein concentration); group B is the neck subcutaneous injection of the anti-inhibin alpha nano antibody Nb-4 recombinant protein (the two immunization doses are 1 mL/piece, and the protein concentration is 0.1 mg/mL); group C was injected subcutaneously into the neck of normal saline (1 mL/dose for both immunizations). After the immunization, the content of reproductive hormone in serum is detected by an enzyme-linked immunosorbent assay, wherein the content of reproductive hormone comprises 6 kinds of hormone including Follicle Stimulating Hormone (FSH), inhibin A (INHA), inhibin B (INHB), estrogen (E2), progestogen (PROG) and Luteinizing Hormone (LH), and the detection results are shown in figures 27-32.

The ELISA genital hormone detection kit is used for respectively detecting the FSH, INHA, INHB, E, P4 and LH contents in the serum of A, B, C groups of sheep before immunization, and the results show that the 6 hormone content differences among A group, B group and C group before immunization have no obvious difference and the average value is close. There was a significant difference in INHA and INHB levels in groups a and B after immunization compared to the control group, and there was a downward trend after immunization, indicating that inhα and Nb-4 immunization both reduced INHA and INHB levels (fig. 27-28); in addition, the FSH levels in group B after immunization were significantly different from those in the control group and tended to increase after immunization (fig. 29), indicating that Nb-4 immunization increased FSH levels, whereas the FSH levels in group B after immunization were not significantly different, but had some ability to increase FSH levels.

The results show that 4 INH alpha specific nano antibodies are successfully screened, and the 4 nano antibodies have better affinity with INH alpha protein, wherein Nb-4 is optimal. The nanobody Nb-4 immunized sheep has obvious inhibition effect on INHA and INHB in sheep body and has promotion effect on FSH level.

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 (8)

1. Nanobody targeting inhα, wherein the nanobody is at least one of Nb-4, nb-15, nb-26 and Nb-57; the amino acid sequences of Nb-4, nb-15, nb-26 and Nb-57 are respectively shown as SEQ ID NO.1-SEQ ID NO. 4.

2. A gene encoding the nanobody of claim 1, wherein the nucleotide sequences of the genes encoding Nb-4, nb-15, nb-26 and Nb-57 nanobodies are shown in SEQ ID No.5-SEQ ID No.8, respectively.

3. An expression vector capable of expressing the gene of claim 2.

4. The expression vector of claim 3, wherein the expression vector is pET-32a.

5. A host cell comprising the expression vector of claim 3 or 4.

6. The host cell of claim 5, wherein the host cell is E.coli BL21 (DE 3).

7. Use of a nanobody according to claim 1 or a gene according to claim 2 or an expression vector according to any one of claims 3 to 4 or a host cell according to any one of claims 5 to 6 for the preparation of an anti-follistatin immune formulation.

8. Use of a nanobody according to claim 1 or a gene according to claim 2 or an expression vector according to any one of claims 3 to 4 or a host cell according to any one of claims 5 to 6 for increasing lambing rate in sheep.