Method for PCR (Polymerase Chain Reaction) amplification of oligopeptide sequence multiplication and high-efficiency expression and purification of prokaryotic protein of oligopeptide sequence multiplication

Abstract

The invention discloses an oligopeptide sequence multiplication PCR (Polymerase Chain Reaction) amplification method and a prokaryotic protein high-efficiency expression and purification method thereof. The method provided by the invention comprises the following steps: A1) designing and synthesizing an amplification template and a primer; the first round of amplification primer pair is composed of a primer F1 and a primer R1; the length of the primer F1 and the length of the primer R1 are both 1.2-1.8 times that of the coding nucleic acid of the oligopeptide; the second-round amplification primer pair is composed of a primer F2 and a primer R2; the length of the primer F2 and the length of the primer R2 are both 1.2-1.8 times that of the coding nucleic acid of the oligopeptide; a2) carrying out first-round PCR amplification to realize oligopeptide coding nucleic acid multiplication or oligopeptide coding nucleic acid repeated series PCR; through a PCR amplification design thought of oligopeptide sequence multiplication, not only can GnRH30 be amplified, but also GnRH50 can be successfully amplified, which cannot be achieved by previous research.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and genetic engineering, and relates to a method for PCR amplification of oligopeptide sequence doubling, high-efficiency expression and purification of prokaryotic proteins. Background Art

[0002] In the fields of molecular biology and genetic engineering, the high-efficiency expression and purification of recombinant proteins are important topics in biomedicine and biotechnology. Especially during the expression process of oligopeptide proteins, how to enhance the immunogenicity of oligopeptide antigens, precisely construct repetitive oligopeptide sequences, and effectively express these sequences remains one of the current technical difficulties. Generally, oligopeptides consist of multiple short amino acid sequences, and the precise replication and efficient tandem construction of these short fragments become a crucial step in the expression system.

[0003] Currently, for the repetitive construction of oligopeptide sequences, traditional PCR amplification techniques often face problems of low efficiency and high error rates when dealing with short DNA sequences. Especially for the amplification of DNA fragments containing repetitive sequences, the specificity and stability of the PCR reaction are further reduced, easily leading to quality problems of the amplification products. This makes it difficult to ensure the amplification accuracy and stability when constructing oligopeptide sequences with high repetition times and long tandem repeats. In addition, existing technologies generally rely on chemical synthesis or high-cost molecular splicing techniques using restriction enzyme cleavage sites to construct repetitive sequences. These methods are not only complex to operate but also costly, making it difficult to meet the requirements of large-scale production and high-efficiency expression.

[0004] For example, GnRH oligopeptide is a monomeric peptide composed of 10 amino acid residues, which has low immunogenicity. To improve its immunogenicity, multiple oligopeptide GnRHs need to be tandemly connected to obtain a polypeptide tandem. Currently, there are GnRH tandems with two or four repeats. The more GnRHs are tandemly connected, the better the immunogenicity of the protein. A common strategy is to tandemly connect GnRH sequences multiple times to enhance the immune response by increasing the number of GnRH insertions. However, due to the limitations of traditional PCR amplification techniques, the successful tandem connection of GnRH sequences multiple times is restricted.

[0005] Therefore, the current research focus is on how to prepare oligopeptides with high repetition times and long tandem repeats. Summary of the Invention

[0006] The technical problem solved by the present invention is how to construct a composite peptide with tandem oligopeptides, specifically how to prepare long tandem oligopeptides by PCR method.

[0007] To solve the above technical problems, the first aspect of the present invention provides a method for doubling oligopeptide-encoding nucleic acids or a PCR method for repetitive tandem of oligopeptide-encoding nucleic acids, including the following steps:

[0008] A1) Design and synthesize amplification templates and primers;

[0009] The amplification template includes template 1 and template 2; the nucleotide sequence of template 1 includes the coding nucleic acid sequence of the oligopeptide; the nucleotide sequence of template 2 includes the reverse complementary sequence of the coding nucleic acid of the oligopeptide;

[0010] The primer includes the first-round amplification primer pair;

[0011] The first-round amplification primer pair consists of primer F1 and primer R1;

[0012] The lengths of primer F1 and R1 are both 1.2 - 1.8 times that of the coding nucleic acid of the oligopeptide;

[0013] Primer F1 includes the coding nucleic acid of the oligopeptide and a partial coding nucleic acid of the oligopeptide from the 5'-end; the partial coding nucleic acid of the oligopeptide consists of the first 40 - 60% of the nucleotide residues of the coding nucleic acid of the oligopeptide from the 5'-end;

[0014] Primer R1 includes the reverse complementary sequence of the coding nucleic acid of the oligopeptide and the reverse complementary sequence of another partial coding nucleic acid of the oligopeptide from the 5'-end; the other partial coding nucleic acid of the oligopeptide consists of the first 40 - 60% of the nucleotide residues of the coding nucleic acid of the oligopeptide from the 3'-end;

[0015] A2) Using template 1 and template 2 as templates, perform the first-round PCR amplification with primer F1 and R1 to obtain the first-round amplification product, realizing the PCR for doubling the coding nucleic acid of the oligopeptide or repeating and concatenating the coding nucleic acid of the oligopeptide.

[0016] In the method described above, the following steps may also be included: recovering the target doubling coding nucleic acid of the oligopeptide or the repeating and concatenating coding nucleic acid of the oligopeptide from the first-round amplification product.

[0017] In the method described above, the enzymes required for the first-round PCR amplification are all Pfu enzyme and Ex enzyme.

[0018] In the method described above,

[0019] The amplification template includes template 1 and template 2;

[0020] The nucleotide sequence of template 1 is, from the 5'-end, successively the coding nucleic acid of the oligopeptide and the codon AAA of lysine Lys; the nucleotide sequence of template 2 is the reverse complementary sequence of template 1;

[0021] The primer includes the first-round amplification primer pair and the second-round amplification primer pair;

[0022] The first-round amplification primer pair consists of primer F1 and primer R1;

[0023] The lengths of the primers F1 and R1 are both 1.2 - 1.8 times the coding nucleic acid of the oligopeptide, and further, the lengths of the primers F1 and R1 are both specifically about 1.5 times the coding nucleic acid of the oligopeptide;

[0024] The primer F1 includes, from the 5'-end, the coding nucleic acid of the oligopeptide, the codon AAA of lysine Lys, and a partial coding nucleic acid of the oligopeptide; the partial coding nucleic acid of the oligopeptide consists of 40 - 60% of the nucleotide residues from the 5'-end of the coding nucleic acid of the oligopeptide (specifically, the partial coding nucleic acid of the oligopeptide may consist of 16 nucleotide residues from the 5'-end of the coding nucleic acid of the oligopeptide);

[0025] The primer R1 includes, from the 5'-end, the reverse complementary sequence of the codon AAA of lysine Lys, the reverse complementary sequence of the coding nucleic acid of the oligopeptide, the reverse complementary sequence of the codon AAA of lysine Lys, and the reverse complementary sequence of another partial coding nucleic acid of the oligopeptide; the other partial coding nucleic acid of the oligopeptide consists of 40 - 60% of the nucleotide residues from the 3'-end of the coding nucleic acid of the oligopeptide (specifically, the other partial coding nucleic acid of the oligopeptide may consist of the first 14 nucleotide residues from the 3'-end of the coding nucleic acid of the oligopeptide).

[0026] In a second aspect, the present invention provides a method for preparing a composite peptide obtained by concatenating multiple oligopeptides, comprising the following steps:

[0027] B1) Design and synthesize an amplification template and primers;

[0028] The amplification template is the amplification template described in the first aspect;

[0029] The primers include the first-round amplification primer pair and the second-round amplification primer pair described in the first aspect;

[0030] The second-round amplification primer pair consists of primer F2 and primer R2;

[0031] The primer F2 includes, from the 5'-end, a restriction enzyme recognition site and the partial coding nucleic acid of the oligopeptide;

[0032] The primer R2 includes, from the 5'-end, another restriction enzyme recognition site, a stop codon, and the reverse complementary sequence of the other partial coding nucleic acid of the oligopeptide;

[0033] B2) Using the template 1 and the template 2 as templates, perform the first-round PCR amplification with the primer F1 and the primer R1 to obtain the first-round amplification product;

[0034] B3) Using the first-round amplification product as a template, perform a second-round PCR amplification with the primer F2 and the primer R2 to obtain a second-round amplification product introducing a restriction enzyme recognition site, and then recover the product in the second-round amplification product that conforms to the coding nucleic acid size of the composite peptide to obtain the coding nucleic acid of the composite peptide;

[0035] B4) Construct the coding nucleic acid of the composite peptide into a prokaryotic expression vector through the introduced restriction enzyme site to obtain a recombinant plasmid; then transfer the recombinant plasmid into Escherichia coli to obtain a recombinant bacterium; induce and culture the recombinant bacterium with IPTG, and collect the induced culture product, namely the composite peptide; the composite peptide includes multiple tandem oligopeptides.

[0036] In the method described above, in the primer F2, a nucleic acid sequence for a purification tag is further included between the restriction enzyme recognition site and the coding nucleic acid of the oligopeptide part;

[0037] In the primer R2, a reverse complementary sequence of the nucleic acid sequence for the purification tag is further included between the stop codon and the reverse complementary sequence of the coding nucleic acid of the other part of the oligopeptide.

[0038] In the method described above, the tag for purification can specifically be a His tag.

[0039] In the method described above, the oligopeptide is GnRH;

[0040] And / or, the composite peptide is GnRHn, where n represents the number of tandem GnRHs. Specifically, GnRHn includes n tandem GnRHs.

[0041] In the above, an amino acid residue can be introduced between every two tandem GnRHs. The amino acid residue can specifically be Lys.

[0042] In the method described above, the second-round amplification primer pair consists of the primer F2 and the primer R2;

[0043] The primer F2 includes, from the 5'-end, a protection base, a restriction enzyme recognition site, a His tag coding nucleic acid, and the coding nucleic acid of the oligopeptide part (specifically, the coding nucleic acid of the oligopeptide part can be 17 nucleotide residues from the 5'-end of the coding nucleic acid of the oligopeptide GnRH);

[0044] The primer R2 includes, from the 5'-end, a protection base, another restriction enzyme recognition site, a stop codon, a reverse complementary sequence of the His tag coding nucleic acid, a reverse complementary sequence of the codon AAA of lysine Lys, and a reverse complementary sequence of the coding nucleic acid of the other part of the oligopeptide (specifically, the coding nucleic acid of the other part of the oligopeptide can be 13 nucleotide residues from the 3'-end of the coding nucleic acid of the oligopeptide GnRH).

[0045] In the method described above,

[0046] In step B4), the conditions for IPTG induction culture are 0.01 - 1.5 mmol / L IPTG, induced expression at 25 - 37 °C for 6 - 8 h;

[0047] Or, in step B4), the conditions for IPTG induction culture are 0.3 mmol / L IPTG, induced expression at 37 °C for 8 h.

[0048] In the method described above,

[0049] In step B4), it further includes the steps of ultrasonic disruption and purification of the induced culture product to obtain the target composite peptide;

[0050] Or, n in GnRHn is greater than or equal to 30;

[0051] Or the composite peptide is GnRH30.

[0052] In the method described above,

[0053] The nucleotide sequence of template 1 is sequence 1;

[0054] Or, the nucleotide sequence of template 2 is sequence 2;

[0055] Or, the nucleotide sequence of primer F1 is sequence 3;

[0056] Or, the nucleotide sequence of primer R1 is sequence 4;

[0057] Or, the nucleotide sequence of primer F2 is sequence 5;

[0058] Or, the nucleotide sequence of primer R2 is sequence 6.

[0059] In the method described above,

[0060] The enzymes required for the first-round PCR amplification and the second-round PCR amplification are both Pfu enzyme and Ex enzyme.

[0061] In a third aspect, the present invention provides the application of the method described in the first aspect, the amplification template and the primer described in the first aspect in the PCR amplification of multiple repeated short fragments or sequence tandem;

[0062] Or preparing a multiplexed oligopeptide-encoding nucleic acid or a repeated tandem oligopeptide-encoding nucleic acid by the method described in the first aspect;

[0063] Or the application of the method described in the second aspect, the amplification template and the primer described in the second aspect in the preparation of a composite peptide obtained by tandem connection of multiple oligopeptides;

[0064] Or a method for preparing a nucleic acid encoding a composite peptide obtained by concatenating multiple oligopeptides, comprising the steps B1-B3 in the method described in the second aspect;

[0065] Or a nucleic acid encoding a composite peptide prepared by the method described in the second aspect;

[0066] Or a composite peptide prepared by the method described in the second aspect.

[0067] The present invention proposes an innovative design concept for PCR amplification technology. It uses special primer design to amplify short DNA sequences and realizes the efficient multiplication of oligopeptide sequences through a prokaryotic expression system. Specifically, the present invention takes the multiplication technology of the mature GnRH decapeptide oligopeptide as a demonstration technology, obtains a composite peptide with 30 tandem repeats of this oligopeptide, designs a short DNA fragment monomer containing 30 bases encoding the mature GnRH decapeptide, and precisely concatenates these fragments through special primer design by PCR technology, multiplying its molecular weight to 30 times the original. This technology not only solves the problems of short fragment amplification and repetitive sequence construction, but also can efficiently express these tandem oligopeptide sequences through a prokaryotic expression system.

[0068] The innovation of this technology lies in that by using special primer design, it successfully overcomes the problem of repetitive sequence amplification in PCR amplification. By precisely controlling the PCR reaction conditions, the target DNA fragment with multiple repeating monomers can be amplified efficiently and precisely, providing a solid foundation for subsequent protein expression. More importantly, through the prokaryotic expression system, these high molecular weight oligopeptide sequences can be efficiently expressed, providing potential technical support for fields such as biomedicine, vaccine research and development, and immunotherapy. This technology can not only be used to enhance the immunogenicity of small molecule oligopeptide antigens, but also be used to identify the binding sites of transcription factors in the promoter region of minor genes and the action sites of miRNA seed sequences binding to target genes, with very wide applications.

[0069] This new design concept for PCR amplification solves the technical problems in repetitive sequence amplification, can efficiently and precisely increase the molecular weight of small molecule oligopeptide sequences, increase their immunogenicity, and provides effective technical support for fields such as biopharmaceuticals, vaccine development, and immunotherapy.

[0070] The present invention adopts an innovative short peptide sequence tandem PCR technology and successfully synthesizes the (GnRH-Lys)30 sequence (abbreviated as the composite peptide GnRH30). This sequence consists of 30 GnRH decapeptide units in the GnRH decapeptide, and each GnRH decapeptide is connected by a lysine (Lys) residue to form a highly repetitive tandem structure. Compared with the previous research method where the maximum repetition of GnRH was 16, the (GnRH-Lys)30 sequence not only significantly increases the tandem number of GnRH but also changes the conformation of the GnRH molecule, thereby maximizing the immunogenicity of the antigen. Subsequently, this sequence was inserted into the pET-32a vector, and recombinant protein was expressed using Escherichia coli BL21(DE3). After sufficient emulsification with MONTANIDE TM ISA 201, the effects of (GnRH-Lys)30 recombinant protein vaccines at different concentrations on the testis, sex hormones, and sexual behavior were evaluated in sexually mature male SD rats. The multi-tandem GnRH antigen has a stronger immunocastration effect. Through the PCR amplification design idea of oligopeptide sequence multiplication, not only GnRH30 can be amplified, but GnRH50 can also be successfully amplified, which could not be achieved in previous studies. Description of the Drawings

[0071] Figure 1 Schematic diagram of the nucleic acid fragment encoding GnRH30.

[0072] Figure 2 Design idea and amplification principle of the nucleic acid primer encoding GnRH30.

[0073] Figure 3 Identification of the 1.5% agarose gel electrophoresis of the first-round PCR product of GnRHn; M1: 100bp Marker, 1: First-round PCR product of GnRHn, M2: 1kb Marker.

[0074] Figure 4 Identification of the 1.5% agarose gel electrophoresis of the second-round PCR product of GnRHn; M: 100bp Marker, 1; 2: Second-round PCR product of GnRHn.

[0075] Figure 5 Identification of the 1.5% agarose gel electrophoresis of the gel recovery product of the second-round PCR of GnRH30; M1: 100bp, Marker1: Second-round PCR product of GnRH30 (red frame area), M2: 1kb Marker.

[0076] Figure 6 Double digestion detection of pET-32a; M1, M2: 1kb Marker, lanes 1 and 3 are pET-32a plasmids, and lanes 2 and 4 are pET-32a double digested.

[0077] Figure 7 DNA sequencing result of GnRH30 recombinant peptide.

[0078] Figure 8 Double digestion detection of pET-32a and pET-32a-GnRH30; M: 1kb Marker, 1: pET-32a plasmid, 2: pET-32a-GnRH30 recombinant plasmid, 3: Double digestion of pET-32a-GnRH30, 4: Single digestion of pET-32a-GnRH30 with EcoRⅤ, 5: Single digestion of pET-32a-GnRH30 with XhoⅠ.

[0079] Figure 9 Detection of the expression forms of the empty plasmid (pET-32a) and the recombinant plasmid (pET-32a-GnRH30); M: PageRuler Protein Ladder 10-180KDa, 1: Supernatant of the bacteria before induction with the empty plasmid, 2: Precipitation of the bacteria before induction with the empty plasmid, 3: Supernatant of the bacteria after induction with the empty plasmid, 4: Precipitation of the bacteria after induction with the empty plasmid, 5: Supernatant of the bacteria before induction with the recombinant plasmid, 6: Precipitation of the bacteria before induction with the recombinant plasmid, 7: Supernatant of the bacteria after induction with the recombinant plasmid, 8: Precipitation of the bacteria after induction with the recombinant plasmid.

[0080] Figure 10 Effect of IPTG concentration on the induced expression of pET32a-GnRH30; M: PageRuler Protein Ladder 10-180KDa, 1: Supernatant of the bacteria after induction with 0.01 mM / L IPTG, 2: Supernatant of the bacteria after induction with 0.3 mM / L IPTG, 3: Supernatant of the bacteria after induction with 0.5 mM / L IPTG, 4: Supernatant of the bacteria after induction with 1 mM / L IPTG, 5: Supernatant of the bacteria after induction with 1.50 mM / L IPTG, 6: Supernatant of the bacteria after induction with 3 mM / L IPTG, 7: Precipitation of the bacteria after induction with 0.01 mM / L IPTG, 8: Precipitation of the bacteria after induction with 0.3 mM / L IPTG, 9: Precipitation of the bacteria after induction with 0.5 mM / L IPTG, 10: Precipitation of the bacteria after induction with 1 mM / L IPTG, 11: Precipitation of the bacteria after induction with 1.50 mM / L IPTG, 12: Precipitation of the bacteria after induction with 3 mM / L IPTG.

[0081] Figure 11To show the effect of time on the induced expression of pET32a-GnRH30; M: PageRuler Protein Ladder 10-180Kda, 1: IPTG induction for 2 h, 2: IPTG induction for 4 h, 3: IPTG induction for 6 h, 4: IPTG induction for 8 h, 5: IPTG induction for 10 h, 6: IPTG induction for 12 h, 7: IPTG induction for 14 h, 8: IPTG induction for 16 h, 9: IPTG induction for 24 h.

[0082] Figure 12 To show the effect of culture temperature on the induced expression of pET32a-GnRH30; M: PageRuler Protein Ladder 10-180KDa, 1: supernatant of cultured bacteria at 16°C, 2: precipitate of cultured bacteria at 16°C, 3: supernatant of cultured bacteria at 25°C, 4: precipitate of cultured bacteria at 25°C, 5: supernatant of cultured bacteria at 37°C, 6: precipitate of cultured bacteria at 37°C

[0083] Figure 13 SDS-PAGE analysis for purifying GnRH30 protein; M: PageRuler Protein Ladder 10-180KDa, 1: precipitate after ultrasonic disruption of induced bacteria, 2: protein band after purification by nickel column.

[0084] Figure 14 Results of single Ex enzyme PCR reaction, M1 is 100bp Marker (Takara, 3422A), M2 is 1kb Marker (Takara, 3426A), 1 is the product of the first round of GnRH PCR.

[0085] Figure 15 Results of single LA enzyme PCR reaction, M1 is 100bp Marker (Takara, 3422A), M2 is 1kb Marker (Takara, 3426A), 1 is the product of the first round of GnRH PCR, 2 is the product of the first round of GnRH PCR.

[0086] Figure 16 Single Pfu enzyme PCR reaction system, M1 is 100bp Marker (Takara, 3422A), M2 is 1kb Marker (Takara, 3426A), 1 is the product of the first round of GnRH PCR, 2 is the product of the first round of GnRH PCR.

[0087] Figure 17 For double LA enzyme + Ex enzyme, M1 is 100bp Marker (Takara, 3422A), M2 is 1kb Marker (Takara, 3426A), 1 is the product of the first round of GnRH PCR.

[0088] Figure 18 SDS-PAGE analysis results of purified GnRH30 protein under different induction conditions; M: Protein Marker; Lane 1: 0.01 mmol / L IPTG, induced at 37°C for 6 h; Lane 2: 0.3 mmol / L IPTG, induced at 16°C for 6 h; Lane 3: 0.3 mmol / L IPTG, induced at 25°C for 6 h; Lane 4: 0.3 mmol / L IPTG, induced at 37°C for 4 h; Lane 5: 0.3 mmol / L IPTG, induced at 37°C for 6 h; Lane 6: 0.3 mmol / L IPTG, induced at 37°C for 8 h;

[0089] Figure 19 BCA protein quantification standard curve; X-axis: Protein concentration (ug / ml); Y-axis: Protein OD562 value.

[0090] Figure 20 Total ion chromatogram of GnRH30 recombinant protein by LC-MS / MS; Horizontal axis: Retention time (min); Vertical axis: Ion intensity (Intensity).

[0091] Figure 21 Second-level mass spectrum of GnRH30 recombinant protein by LC-MS / MS; Abscissa: Mass-to-charge ratio (m / z); Ordinate: Ion intensity (%).

[0092] Figure 22 Western blot analysis results of GnRH30 recombinant protein; Lane M: Protein Marker; Lane 1: Recombinant protein GnRH30. Specific Embodiments

[0093] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0094] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0095] Unless otherwise specified, in the following embodiments, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0096] Example 1. PCR amplification of nucleic acid encoding composite peptide GnRHn containing multiple tandem repeated GnRH coding sequences

[0097] The natural oligopeptide GnRH is a decapeptide sequence, specifically: Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (QHWSYGLRPG, Sequence 7), Glutamic acid-Histidine-Tryptophan-Serine-Tyrosine-Glycine-Leucine-Arginine-Proline-Glycine. The nucleotide sequence of the nucleic acid encoding the oligopeptide GnRH is: CAGCACTGGTCCTATGGACTGCGCCCTGGA.

[0098] The composite peptide GnRHn refers to a composite peptide including n tandem oligopeptide GnRHs, where n is greater than or equal to 30.

[0099] In the design of GnRHn, a lysine Lys is inserted as a linker peptide between every two tandem oligopeptide GnRHs, which not only realizes the effective connection between oligopeptides but also makes the composite peptide more easily recognized by the body's immune system by changing the natural conformation, thereby improving the overall immunogenicity. To improve the purification efficiency, 6×His Tag labels are connected to both ends of GnRHn, facilitating the capture of the target protein by nickel columns in the later stage. At the same time, specific restriction enzyme sites (EcoRⅤ and XhoⅠ) are designed at both ends of the GnRHn gene for the construction of prokaryotic expression vectors in the later stage.

[0100] Taking the composite peptide GnRH30 composed of 30 tandem oligopeptide GnRHs as an example, the schematic diagram of the nucleic acid encoding the composite peptide GnRHn is as Figure 1 shown. The nucleic acid encoding the composite peptide GnRH30 includes the EcoRⅤ restriction enzyme recognition site, the 6×His Tag label nucleic acid sequence, the nucleic acid sequences of the 1st GnRH (denoted as GnRH1 in the figure) to the 30th GnRH (denoted as GnRH30 in the figure), the 6×His Tag label nucleic acid sequence, the stop codon, and the XhoⅠ restriction enzyme recognition site; and 1 lysine Lys codon (denoted as Lys-C in the figure) is inserted between every two GnRH nucleic acid sequences. The nucleotide sequence of the composite peptide GnRH30 is as shown in Sequence 8.

[0101] I. Primer Design

[0102] Using the nucleic acid sequence encoding the oligopeptide GnRH, a template for synthesizing the nucleic acid encoding the composite peptide GnRHn was designed, which includes GnRH template+ and GnRH template- shown in Table 1 as amplification templates. At the same time, 2 pairs of specific primers GnRH F1, GnRH R1, GnRH F2, and GnRH R2 (shown in Table 1) were designed for the two-round PCR amplification of the composite peptide GnRHn.

[0103] Table 1 shows the amplification templates and primer sequences of the composite peptide GnRHn

[0104]

[0105] Among the above,

[0106] GnRH template+ is the coding nucleic acid of the oligopeptide GnRH, and the codon AAA (in bold red font) of lysine Lys is introduced at its 3' end; if lysine Lys is not required in later experiments, the codon of lysine Lys can also not be added here.

[0107] GnRH template- is the reverse complementary sequence of GnRH template+.

[0108] GnRH F1 and GnRH R1 are the forward and reverse primers for the first-round PCR amplification.

[0109] The GnRH F1 primer includes the coding nucleic acid of the oligopeptide GnRH, the codon AAA (in bold red font) of lysine Lys, and 16 nucleotide residues from the 5' end of the coding nucleic acid of the oligopeptide GnRH; the size of the GnRH F1 primer is about 1.5 times that of the coding nucleic acid of the oligopeptide GnRH.

[0110] The GnRH R1 primer includes the reverse complementary sequence of the codon AAA (in bold red font) of lysine Lys, the reverse complementary sequence of the coding nucleic acid of the oligopeptide GnRH, the reverse complementary sequence of the codon AAA of lysine Lys, and the reverse complementary sequence of 14 nucleotide residues from the 3' end of the coding nucleic acid of the oligopeptide GnRH; the size of the GnRH R1 primer is about 1.5 times that of the GnRH gene of the oligopeptide.

[0111] The GnRH F2 primer includes a protective base (italic), an EcoRⅤ restriction enzyme recognition site (underlined solid line), a His-tag coding nucleic acid (underlined wavy line), and 17 nucleotide residues from the 5' end of the coding nucleic acid of the oligopeptide GnRH;

[0112] The GnRH R2 primer includes a protective base (italic), an XhoⅠ restriction enzyme recognition site (underlined solid line), a stop codon (underlined ellipsis), the reverse complementary sequence of the His-tag coding nucleic acid (underlined wavy line), the reverse complementary sequence of the codon AAA of lysine Lys (in bold red font), and the reverse complementary sequence of 13 nucleotide residues from the 3' end of the coding nucleic acid of the oligopeptide GnRH.

[0113] Among them, two His-tags upstream and downstream in the amplified fragment of the GnRH F2 primer and GnRH R2 primer will greatly improve the purification efficiency of the later protein; the restriction enzyme digestion sites in the amplified fragment are used to ligate the target sequence into the prokaryotic expression vector pET-32a(+).

[0114] II. Preparation of the composite peptide GnRH30 by PCR amplification

[0115] 1. First-round PCR amplification of GnRHn tandem repeat fragments

[0116] The design concept and schematic diagram of the first-round PCR are as Figure 2 shown.

[0117] Using the synthesized GnRH template+ and GnRH template- in the above item 1 as templates, and GnRH F1 and GnRH R1 as primers, the PCR reaction conditions are: 94°C for 30 s; 50°C for 30 s; 72°C for 30 s, for a total of 10 cycles, 94°C for 30 s; 55°C for 30 s; 72°C for 30 s, for a total of 30 cycles, 72°C for 10 min, and stored at 4°C. The PCR reaction system is shown in Table 2, and the first-round PCR amplification is carried out.

[0118] GnRH fragments with different tandem numbers (n) are obtained through the first-round PCR amplification reaction. After detection by 1.5% agarose gel electrophoresis, the results are as Figure 3 shown. It can be seen that the obtained product is a smear band. The first-round PCR product is recovered and purified using an agarose gel recovery kit.

[0119] Table 2 is the first-round PCR reaction system of GnRHn

[0120] Reactant Name Volume (μL) GnRH template+ 0.25 GnRH template- 0.25 GnRH F1 (10 μmol / L) 1.25 GnRH R1 (10 μmol / L) 1.25 10×Ex Taq Buffer 2.5 dNTP Mixture (2.5 mM each) 2 Pfu DNA polymerase (Sangon, B600003) 0.5 Ex Taq (Takara, RR001) 0.3 <![CDATA[ddH2O]]> Up to 25

[0121] 2. Second-round PCR amplification of composite peptide GnRHn

[0122] Using the PCR product recovered from the gel in the above item 1 as a template, and GnRH F2 and GnRH R2 as primers, the PCR reaction conditions are: 94°C for 30 s; 55°C for 30 s; 72°C for 30 s, for a total of 10 cycles, 94°C for 30 s; 60°C for 30 s; 72°C for 30 s, for a total of 30 cycles, 72°C for 10 min, and stored at -20°C. The PCR reaction system is shown in Table 3, and the second-round PCR amplification of GnRHn is carried out.

[0123] The second-round PCR product of GnRHn-encoded nucleic acid is detected by 1.5% agarose gel electrophoresis. The results are as Figure 4 shown. It can be seen that the product is a smear band.

[0124] Table 3 is the second-round PCR reaction system of GnRHn

[0125] Reactant Name Volume (μL) GnRHn First Round PCR Recovery Product 0.5 GnRH F2 (10 μmol / L) 1.25 GnRH R2 (10 μmol / L) 1.25 10×Ex Taq Buffer 2.5 dNTP Mixture (2.5 mM each) 2 Pfu DNA polymerase (Sangon, B600003) 0.5 Ex Taq (Takara, RR001) 0.3 <![CDATA[ddH2O]]> Up to 25

[0126] 3. Gel recovery of GnRH30 product

[0127] To prepare the polypeptide tandem complex peptide GnRH30, only the coding DNA fragments of 30 oligopeptides GnRH need to be recovered, and the fragment size is about 1000 bp.

[0128] Select 1Kb and 100bp DNA ladders to accurately locate the region near 1Kb in the GnRHn second-round PCR product for recovery. The recovery schematic diagram is as follows Figure 5 As shown in the red box.

[0129] 4. Verification of the recovered product

[0130] Send the recovered product obtained in 3 to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing. After sequencing verification, it was found that the full length of the fragment recovered in the region near 1Kb is 1029 bp (Sequence 8), which contains 30 GnRH coding nucleic acids, and Lys codons are connected between each two. Therefore, this fragment is named the coding nucleic acid of the complex peptide GnRH30.

[0131] Example 2. Optimization of amplification conditions

[0132] After multiple condition explorations and optimizations of the GnRHn first-round PCR reaction system in Table 2 of Example 1, the purpose is to obtain a Smear band of the expected target (containing about 1000 bp). The specific experimental scheme for exploring the types of enzymes required for amplification is as follows:

[0133] Single Ex enzyme GnRHn first-round PCR reaction system (25 μL): 0.25 μL each of GnRH template+ and GnRH template-, 1.25 μL each of GnRH F1 and GnRH R1, 2.5 μL of 10×Ex Taq Buffer, 2 μL of dNTP Mixture (2.5 mM each), 0.5 μL of Ex Taq, 17 μL of ddH2O.

[0134] Single LA enzyme PCR reaction system (25 μL): 0.25 μL each of GnRH template+ and GnRH template-, 1.25 μL each of GnRH F1 and GnRH R1, 2.5 μL of 10×LA Taq Buffer, 2 μL of dNTP, 0.5 μL of LA Taq, 17 μL of ddH2O.

[0135] Single Pfu enzyme PCR reaction system (25 μL): 0.25 μL each of GnRH template+ and GnRH template-, 1.25 μL each of GnRH F1 and GnRH R1, 2.5 μL of 10×Pfu Taq Buffer, 2 μL of dNTP, 0.5 μL of Pfu Taq, 17 μL of ddH2O.

[0136] Double LA enzyme + Ex enzyme PCR reaction system (25 μL): 0.25 μL each of GnRH template+ and GnRH template-, 1.25 μL each of GnRH F1 and GnRH R1, 2.5 μL of 10× LA Buffer, 2 μL of dNTP, 0.5 μL of LA enzyme, 0.3 μL of Ex Taq, 16.7 μL of ddH2O.

[0137] PCR reaction conditions: 94°C for 30 s; 50°C for 30 s; 72°C for 30 s, a total of 10 cycles, 94°C for 30 s; 55°C for 30 s; 72°C for 30 s, a total of 30 cycles, 72°C for 10 min, stored at 4°C.

[0138] The first-round PCR products of GnRHn amplified by various enzymes were detected by 1.5% agarose gel electrophoresis, and the results are as Figures 14 - 17 shown. Only the double-enzyme system of Pfu enzyme + Ex enzyme obtained a Smear band of the expected size at around 1000 bp, while Figures 14 - 17 the systems shown did not amplify the expected band at 1000 bp; it indicates that the single-enzyme amplification systems using only Ex enzyme or only Pfu enzyme, or the double-enzyme system of LA enzyme + Ex enzyme, all had worse amplification results than the double-enzyme system of Pfu enzyme + Ex enzyme in Table 2.

[0139] Therefore, the GnRHn first-round PCR reaction system shown in Table 2 of Example 1 is the optimal system. The Pfu enzyme contained in the optimized system can ensure the accuracy of the amplified multiple repeats and reduce the possibility of base mismatch; the Ex enzyme can ensure the smooth amplification of multiple-fold and sufficiently long products, improving the amplification effect, and neither of them can be missing. The difference between the second-round system and the first-round system is only in the template and primers. Therefore, the double-enzyme system of Pfu enzyme + Ex enzyme explored in the first round was also used for the first-round amplification, and the only difference was that the template was replaced with the first-round amplification product.

[0140] Example 2. Expression and purification of composite peptide GnRH30

[0141] I. Construction of pET32a-GnRH30 prokaryotic expression vector

[0142] The coding nucleic acid of the composite peptide GnRH30 recovered from Example 1 II and the prokaryotic expression vector plasmid pET-32a(+) plasmid (P3100, Solarbio, China) were respectively double-digested with the restriction endonucleases EcoRⅤ (Takara, 1042S) and XhoⅠ (Takara, 1094S). After digestion, the target fragments were recovered, and the coding nucleic acid of the composite peptide GnRH30 after digestion and the target of the pET-32a(+) plasmid after digestion were ligated by T4 DNA ligase to construct the recombinant plasmid pET32a-GnRH30.

[0143] 1. Ligation

[0144] 1) Double digestion of pET-32a(+) plasmid and the recovered product of composite peptide GnRH30

[0145] The pET-32a(+) plasmid and the coding nucleic acid of the composite peptide GnRH30 were simultaneously double-digested with the restriction endonucleases EcoRⅤ and XhoⅠ. The reaction system is shown in Table 4 below. After digestion at 37°C for 12 h, the digestion products were detected by 1% agarose gel electrophoresis.

[0146] Table 4 shows the digestion system

[0147] Reactant Name Volume (μl) EcoR Ⅴ (10 IU / μl) 1 Xho Ⅰ (10 IU / μl) 1 10×H Buffer 2 Plasmid / PCR Recovery Product ≤1 μg <![CDATA[ddH2O]]> Up to 20

[0148] The electrophoresis results are as Figure 6 shown. The pET32a vector before digestion presented three molecular forms: covalently closed DNA, linear DNA, and open circular DNA. The backbone fragment of the pET32a vector after digestion was 5851 bp. The large fragment of the pET32a vector after digestion was recovered using the E.Z.N.A. Gel Extraction Kit (Omega, D2500). The specific operation steps were referred to the instruction manual to obtain the large fragment of the pET32a vector.

[0149] Since only the digestion sites were removed after digestion of the coding nucleic acid of the composite peptide GnRH30, the size difference from the original recovered product could not be seen in the agarose gel electrophoresis picture. The product after digestion of the coding nucleic acid of GnRH30 was recovered and denoted as the recovered coding nucleic acid of GnRH30.

[0150] 2) Reaction for ligation of the recombinant plasmid

[0151] The recovered coding nucleic acid of the composite peptide GnRH30 was ligated to the large fragment of the pET32a vector using -T Easy Vector Systems (Promega, A1360), and ligated overnight at 4°C to obtain pET32a-GnRH30. The ligation system is shown in Table 5 below.

[0152] Table 5 shows the connection system

[0153]

[0154] 2. Cloning of recombinant plasmid pET32a-GnRH30

[0155] Take 80 μl of Trans5α Chemically Competent Cell (Tran, CD201) thawed on ice, add 5 μl of the pET32a-GnRH30 ligation product to the competent cells, gently mix and incubate on ice for 30 min, heat shock in a 42 °C water bath for 45 s, incubate on ice for 2 min, then add 500 μL of sterile LB liquid medium (without Amp), mix well and place in a 37 °C incubator, culture at 200 rpm for 1 h to recover Escherichia coli. Take 100 μL of the competent cells and add them to the LB solid medium (containing Amp), spread the cells evenly. Incubate upright in the dark for 1 h, wait for the liquid to be absorbed, invert the culture dish, and culture overnight at 37 °C and 200 rpm to obtain monoclonal colonies transfected with pET-32a-GnRH30. Use the monoclonal colonies transfected with PET-32a(+) as a control.

[0156] 3. Identification of pET32a-GnRH30 positive bacterial liquid by PCR

[0157] Pick the monoclonal colonies transfected with pET-32a-GnRH30 and the monoclonal colonies transfected with pET-32a obtained above and inoculate them into LB liquid medium (containing Amp), culture at 37 °C and 200 rpm for 8 h to obtain the recombinant vector pET-32a-GnRH30 bacterial liquid and the empty vector pET-32a bacterial liquid.

[0158] Use specific bacterial liquid PCR primers (Table 6) to perform PCR verification on the above-mentioned bacterial liquids respectively, and detect the PCR products of the bacterial liquids by 1% agarose gel electrophoresis.

[0159] The PCR reaction program is: pre-denaturation at 94 °C for 5 minutes; then perform 35 cycles, including denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 80 s; finally, extend at 72 °C for another 20 minutes. The PCR reaction system is 25 μL, including 0.5 μL of bacterial liquid, 0.5 μL of F / R primers, 2.5 μL of 10× Buffer, 0.5 μL of dNTP, 0.5 μL of Tag DNA polymerase, 1.5 μL of MgCl2, and 18 μL of dd H2O.

[0160] Table 6 shows the bacterial liquid PCR primers

[0161]

[0162] PCR identification of the empty pET-32a bacterial solution and the recombinant vector pET-32a-GnRH30 bacterial solution showed a specific band at 324 bp and 1332 bp respectively, which was consistent with the expected target fragment size. The positive recombinant vector pET-32a-GnRH30 bacterial solution was obtained.

[0163] The positive recombinant vector pET-32a-GnRH30 bacterial solution was inoculated into 50 ml of sterile LB liquid medium (containing Amp), and cultured overnight at 37 °C and 200 rpm / min. The cultured bacterial solution was sent to Shanghai Jierui Biotechnology Co., Ltd. for sequencing. Figure 7 The sequencing results showed that the coding nucleic acid (1029 bp) of the composite peptide GnRH30 had been successfully inserted into the pET32a vector, indicating that pET32a-GnRH30 was successfully constructed. The His tag in this vector was co-expressed with the coding nucleic acid of the composite peptide GnRH30 to obtain the composite peptide GnRH30 recombinant protein (hereinafter also referred to as the GnRH30 recombinant protein).

[0164] 4. Double digestion identification of the pET32a-GnRH30 recombinant plasmid

[0165] The empty pET-32a bacterial solution and the positive recombinant vector pET-32a-GnRH30 bacterial solution were cultured and then the plasmids were extracted using the E.Z.N.A. Plasmid Mini KitⅡ (Omega, D6945). Then, the pET-32a and pET-32a-GnRH30 plasmids were identified by double digestion with the restriction endonucleases EcoRⅤ and XhoⅠ. The reaction system was as shown in Table 7 below. After digestion at 37 °C for 12 h, the digested products were detected by 1% agarose gel electrophoresis.

[0166] Table 7 is for double digestion verification

[0167] Reactant Name Volume (μl) EcoRⅤ (10 IU / μl) 1 XhoⅠ (10 IU / μl) 1 10×H Buffer 2 pET - 32a / pET - 32a - GnRH30 ≤1 μg <![CDATA[ddH2O]]> Up to 20

[0168] The electrophoresis results were as Figure 8 shown. Before double digestion, both pET-32a and pET-32a-GnRH30 showed three molecular forms: covalently closed DNA, linear DNA, and open circular DNA. After double digestion, pET-32a-GnRH30 had a specific band at 1041 bp (inserted fragment) and 5848 bp (vector backbone) respectively, which was consistent with the expected target fragment size. Both the double digestion and sequencing results indicated that the GnRH30 fragment had been successfully inserted into the pET32a vector, and the recombinant vector pET-32a-GnRH30 was obtained.

[0169] II. Induced expression of the GnRH30 recombinant protein

[0170] 1. Transformation of pET-32a-GnRH30

[0171] Take 50 μl of BL21(DE3) competent cells (Trangenes, CD601) thawed on ice, add 1 μL of pET-32a and pET-32a-GnRH30 plasmids respectively, mix gently and incubate on ice for 30 min. Then perform heat shock at 42 °C for 45 s, incubate on ice for 2 min, add 500 μL of sterile LB liquid medium (without Amp), mix well and place in a 37 °C incubator, culture at 200 rpm / min for 1 h to recover Escherichia coli. Take 100 μL of competent cells and add them onto LB solid medium (containing Amp), spread the cells evenly. Incubate upright for 1 h in the dark until the liquid is absorbed, then invert the culture dish and culture overnight at 37 °C, 200 rpm / min to obtain pET32a bacterial solution (denoted as BL21(DE3) / pET32a) and pET32a-GnRH30 bacterial solution.

[0172] Inoculate the BL21(DE3) / pET32a-GnRH30 bacterial solution into sterile LB liquid medium (containing Amp), culture at 37 °C, 200 rpm / min overnight for expansion and use it for subsequent induction expression.

[0173] 2. Induction expression of GnRH30 recombinant protein

[0174] Take 1 ml of the BL21(DE3) / pET32a-GnRH30 bacterial solution and BL21(DE3) / pET32a respectively and inoculate them into LB medium containing Amp, culture at 37 °C, 200 rpm / min for 2 h. When the OD600 value of the bacterial solution is 0.6, take 1 mL of the bacterial solution as the control before induction of the recombinant vector; for the remaining bacterial solution, add 10 μL of inducer with a concentration of 100 mM Isopropyl β-D-Thiogalactoside (IPTG) (BioFroxx, 122GR005) per 1 mL to make the final concentration of IPTG 1 mM, mix well and continue to induce culture at 37 °C, 200 rpm / min for 8 h as the control after induction of the recombinant vector.

[0175] 3. Ultrasonic disruption of pET32a-GnRH30 protein sample

[0176] After the above-mentioned induced cultures, each bacterial solution was centrifuged at 6,000 rpm / min for 10 min. The supernatant was discarded and the bacterial pellet was retained. Then, 100 μL of lysis buffer was added to every 1 mL of the bacterial pellet: Weigh Tris 50 mM, NaCl 300 mM, and imidazole 10 mM in proportion and place them in a beaker. After volume-fixing with ddH2O, adjust the pH to 8.0. Filter through a 0.22 μm filter, add 1 μL of 100 mmol / L phenylmethanesulfonyl fluoride (PMSF) (Solarbio, P0100), place it in liquid nitrogen for 5 min, then melt it in a water bath at 42 °C, and repeat the freeze-thaw cycle 5 times to lyse the bacteria. To ensure complete lysis of the bacteria, ultrasonic disruption was performed at a power of 30 W with an ultrasonic duration of 5 s and an interval of 6 s, and the total duration was 90 min. To avoid excessive heat generation caused by ultrasound, the bacterial solution must be kept immersed in an ice bath during the ultrasonic process.

[0177] 4. SDS-PAGE Electrophoresis Detection of GnRH30 Recombinant Protein

[0178] After ultrasonic disruption, resuspend the bacterial pellet with Binding Buffer (weigh Tris 20 mM, NaCl 500 mM, imidazole 5 mM, and urea 8 M in proportion and place them in a beaker. After volume-fixing with ddH2O, adjust the pH to 7.9. Filter through a 0.22 μm filter and store at room temperature), mix well to dissolve the inclusion bodies, then filter through a 0.45 μm filter membrane to obtain the inclusion body protein sample, and store it at -20 °C. Take 80 μL of the supernatant and precipitate after disrupting the bacteria respectively, then add 20 μL of protein tracking loading buffer (ComWin Biotech, CW0052), then pipette and mix well and centrifuge briefly, boil in a water bath for 10 min, cool to room temperature, centrifuge at 2,000 rpm / min for 30 s - 60 s, take 20 μL of the supernatant and perform SDS-PAGE electrophoresis using a 5% stacking gel and a 10% separating gel with a SDS-PAGE gel preparation kit (ComWin Biotech, CW0022M), and stain with Coomassie ProteinShow-G250 protein rapid staining reagent (ComWin Biotech, CW0023), and take a photo after decolorizing with the decolorizing solution.

[0179] The electrophoresis results are as Figure 9 shown. There was protein expression in the supernatant sample of the BL21(DE3) / pET32a empty vector after induction at 13.31 KDa, indicating that the empty vector protein mainly exists in the form of soluble protein; there was protein expression in the precipitate sample of the BL21(DE3) / pET32a-GnRH30 recombinant protein after induction at 51.59 KDa (the same size as the target recombinant protein GnRH30), indicating that this recombinant protein mainly exists in the form of inclusion body protein.

[0180] 5. Optimization of the Induced Expression Conditions of pET32a-GnRH30 Protein

[0181] In the previous experiment for determining the protein expression form, it was found that the pET32a-GnRH30 recombinant protein mainly existed in the precipitate in the form of inclusion bodies. To increase the expression level of the target protein, the optimization was carried out from three factors: the concentration of IPTG induction (0.01 mmol / L, 0.3 mmol / L, 0.5 mmol / L, 1.0 mmol / L, 1.5 mmol / L, 3 mmol / L, Figure 10 )), the induction time (2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 24 h Figure 11 ) and the culture temperature (16 °C, 25 °C, 37 °C, Figure 12 ).

[0182] 1) Optimization of the IPTG Concentration for Recombinant Protein Expression

[0183] Pick the positive clone strain BL21(DE3) / pET32a-GnRH30 and inoculate it into the ampicillin-resistant LB liquid medium. Culture it with shaking at 37 °C until the OD600 is about 0.7. Add IPTG with concentrations of 0.01 mmol / L, 0.3 mmol / L, 0.5 mmol / L, 1.0 mmol / L, 1.5 mmol / L, and 3 mmol / L into the bacterial solution respectively, and induce it at 37 °C for 8 hours. After the induction is completed, take the supernatant and precipitate, and after treatment according to the above 3 and 4, perform SDS-PAGE electrophoresis (5% stacking gel and 10% separating gel) for detection and analysis.

[0184] The results are as Figure 10 shown. It can be seen that obvious expression of the target protein can be detected at 0.01 mmol / L IPTG (lane 7). When the IPTG concentration is 0.3 mmol / L (lane 8), the protein expression level reaches the highest level. Continuing to increase the IPTG concentration (0.5 - 3 mmol / L) does not lead to a significant increase in the expression level. Therefore, 0.3 mmol / L IPTG can meet the optimal induction expression requirements of the GnRH30 recombinant protein.

[0185] 2) Optimization of the Expression Time of Recombinant Protein

[0186] According to the method in 1) above, induce the expression of BL21(DE3) / pET32a-GnRH30 with the optimal concentration of 0.3 mmol / L IPTG. The induction time gradients are 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 16 h and 24 h respectively, and the induction temperature is 37 °C. After the induction is completed, take the supernatant and precipitate, and after treatment according to the above 3 and 4, perform SDS-PAGE electrophoresis for detection and analysis.

[0187] The results are as Figure 11 shown. It can be seen that during the initial stage of induction (2 - 6 h), the target protein was expressed (lanes 1 - 3). As the induction time was extended to 8 h, the protein expression level increased significantly (lane 4), and remained at a similar level at subsequent time points (10 - 24 h) (lanes 4 - 9). This indicates that the protein expression reached a saturated level after 8 h of induction, which is the optimal induction time.

[0188] 3) Optimization of the temperature for recombinant protein expression

[0189] According to the method in 1) above, BL21(DE3) / pET32a - GnRH30 was induced to express at the optimal concentration of 0.3 mmol / L IPTG for 8 h, and the induction temperature gradients were 16 °C, 25 °C, and 37 °C respectively. After induction, the supernatant and precipitate were taken, and after treatment according to 3 and 4 above, SDS - PAGE electrophoresis was performed for detection and analysis.

[0190] The results are as Figure 12 shown. It can be seen that the protein expression level at 37 °C (lane 6) was significantly higher than that at 16 °C and 25 °C, and 37 °C is the optimal induction temperature.

[0191] III. Purification of GnRH30 recombinant protein

[0192] 1 mL of the BL21(DE3) / pET32a - GnRH30 bacterial solution was inoculated into LB medium containing Amp and cultured at 37 °C and 200 rpm / min. When the OD600 value of the bacterial solution reached 0.6, different IPTGs were added to make their final concentrations 0.01 or 0.3 mmol / L respectively, and induction expression was carried out at 16 °C, 25 °C, or 37 °C for 4 h, 6 h, or 8 h. The above - mentioned ultrasonic disruption method was used for cell disruption and inclusion body protein sample preparation.

[0193] Subsequently, the inclusion body protein dissolved in Binding Buffer was purified using Ni-agarose gel (ComWin Biotech, CW0010). First, the affinity chromatography column (Solarbio, DS0150) was fixed on a support. After gently shaking and mixing the Ni-agarose gel, it was added to the chromatography column using a pipette (1 ml of nickel was added per 100 ml of bacterial solution), and it was left standing at room temperature for 10 min, then the ethanol was discarded; 5 column volumes of deionized water were added to the loaded column to wash away the residual ethanol, and then the column was equilibrated with 8 column volumes of Binding Buffer, repeating this 2 times; the stock solution of inclusion body protein was loaded onto the column. To ensure sufficient contact between the inclusion body protein solution and the nickel column, the target protein with a 6×His tag was bound to the nickel column. In this experiment, the flow rate was limited to about 10 column volumes per hour by controlling the speed of the added protein stock solution. Subsequently, the column was washed with 15 column volumes of Binding Buffer, repeated 3 times, to wash away the impurity proteins; then 15 ml of Elution Buffer was used to elute the target protein with a 6×His tag, and the elution peak was collected to obtain the purified GnRH30 recombinant protein solution (the solvent was Elution Buffer). Elution Buffer: Weigh Tris 20 mM, NaCl 500 mM, imidazole 500 mM, and urea 8 M in proportion and place them in a beaker. After making up the volume with ddH2O, adjust the pH to 7.9, filter it with a 0.22 μm filter, and store it at room temperature.

[0194] The purified GnRH30 recombinant protein solutions obtained under the above different induction conditions (denoted as GnRH-1 to GnRH-6 shown in Table 8) were subjected to SDS-PAGE electrophoresis using a 5% stacking gel and a 10% separating gel, and the results were as Figure 18 shown. A single main band was visible at 54.41 kDa for the recombinant protein under different induction conditions, which was consistent with the expected molecular weight, and there were almost no impurity protein bands, indicating a significant purification effect. Among them, the band in lane 6 (0.3 mmol / L IPTG, induced at 37 °C for 8 h) showed the darkest color development, indicating that a high concentration of GnRH recombinant protein could be obtained by extending the induction time to 8 h at 37 °C, and the purity of the protein after purification met the requirements for vaccine preparation. In contrast, the band in lane 2 (0.3 mmol / L IPTG, induced at 16 °C for 6 h) was the lightest, indicating that the low temperature significantly inhibited protein expression. The band intensities in lane 1 (0.01 mmol / L IPTG, induced at 37 °C for 6 h), lane 3 (0.3 mmol / L IPTG, induced at 25 °C for 6 h), and lane 4 (0.3 mmol / L IPTG, induced at 37 °C for 4 h) were between those in lane 2 and lanes 5 / 6, suggesting that a lower IPTG concentration (0.01 mmol / L), medium temperature (25 °C), or shorter induction time (4 h) would all reduce the expression level of GnRH protein.

[0195] Therefore, it was finally determined that when the OD600 value of the bacterial solution was 0.6, the final concentration of IPTG was added to 0.3 mmol / L, and induction at 37 °C for 8 h was the optimal induction condition for preparing and purifying the GnRH30 recombinant protein.

[0196] The BCA protein quantification method was used to detect the concentration of the purified GnRH30 recombinant protein solution obtained under different induction conditions (the standard curve is as Figure 19 shown). The results are shown in Table 8. It can be seen that the concentration of the purified GnRH30 recombinant protein solution of GnRH-6 is very high, reaching up to 1439.38 μg / ml at most.

[0197] Table 8 shows the concentration of the GnRH30 recombinant protein solution

[0198]

[0199] In summary, induction with 0.3 mmol / L IPTG at 37 °C for 8 h is the optimal expression condition for the GnRH30 recombinant protein. The purified protein has a high purity and is suitable for subsequent vaccine development research.

[0200] The purified GnRH30 recombinant protein solution obtained by induction with 0.3 mmol / L IPTG at 37 °C for 8 h was subjected to SDS-PAGE electrophoresis detection. The results are as Figure 13 shown. The purified protein band is relatively single, with a size of approximately 51.59 KDa, which meets the expectations, indicating that the expressed product can efficiently bind to the nickel column and the effective purification of the target protein has been successfully achieved.

[0201] IV. Mass Spectrometry Identification

[0202] To identify the expression of the (GnRH) recombinant protein, two techniques, liquid chromatography-tandem mass spectrometry (LC-MS / MS) and Western blot, were used for analysis in this study.

[0203] First, the LC-MS / MS technique was used to identify the purified GnRH30 recombinant protein obtained by induction with 0.3 mmol / L IPTG at 37 °C for 8 h as described above. Its total ion current chromatogram ( Figure 20 ) shows multiple ion peaks, indicating the presence of multiple peptide components in the sample. Further secondary mass spectrometry analysis ( Figure 21 ) provides the fragment ion information of these peptides. By comparing with the theoretical amino acid sequence of the composite peptide GnRH30, the peptide information is completely matched with the expected sequence.

[0204] Subsequently, the purified GnRH30 recombinant protein obtained by inducing with 0.3 mmol / L IPTG at 37°C for 8 hours was immunologically identified using Western blot technology. Rabbit anti-GnRH polyclonal antibody (1:4000, ab189878, Abcam, UK) was used as the primary antibody, and HRP-labeled goat anti-rabbit IgG (1:10000, AS014, ABclonal, China) was used as the secondary antibody for immunodetection. The results are as Figure 22 shown. In the corresponding lane (lane 1), a single target band corresponding to the expected molecular weight was detected, proving that the GnRH30 recombinant protein was successfully expressed and could be specifically recognized by the rabbit anti-GnRH polyclonal antibody, with good specificity and integrity.

[0205] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A method for multiplying an oligopeptide encoding nucleic acid or a PCR method for repeating and tandemly sequencing an oligopeptide encoding nucleic acid, comprising the following steps: A1) Design synthetic amplification templates and primers; The amplification template includes template 1 and template 2; the nucleotide sequence of template 1 includes the nucleic acid sequence encoding the oligopeptide; the nucleotide sequence of template 2 includes the reverse complementary sequence of the nucleic acid encoding the oligopeptide; The primers include a first round amplification primer pair; The first round amplification primer pair consists of primer F1 and primer R1; The lengths of the primers F1 and R1 are both 1.2-1.8 times the length of the nucleic acid encoding the oligopeptide; The primer F1 includes the nucleic acid encoding the oligopeptide and the nucleic acid encoding the oligopeptide portion from the 5' end; the nucleic acid encoding the oligopeptide portion consists of the first 40-60% of the nucleotide residues of the nucleic acid encoding the oligopeptide from the 5' end; The primer R1 includes the reverse complementary sequence of the nucleic acid encoding the oligopeptide and the reverse complementary sequence of the nucleic acid encoding another part of the oligopeptide from the 5' end; the nucleic acid encoding another part of the oligopeptide consists of the first 40-60% of the nucleotide residues of the nucleic acid encoding the oligopeptide from the 3' end; A2) using the template 1 and the template 2 as templates, and using the primers F1 and R1 to perform a first round of PCR amplification to obtain a first round of amplification products, thereby achieving PCR of oligopeptide encoding nucleic acid doubling or oligopeptide encoding nucleic acid repeating in series.

2. The method according to claim 1, characterized in that: The enzymes required for the first round of PCR amplification are Pfu enzyme and Ex enzyme.

3. A method for preparing a composite peptide by connecting multiple oligopeptides in series, comprising the following steps: B1) Design synthetic amplification templates and primers; The amplification template is the amplification template described in claim 1 or 2; The primers include the first round amplification primer pair and the second round amplification primer pair in claim 1 or 2; The second round amplification primer pair consists of primer F2 and primer R2; The primer F2 includes an enzyme recognition site and the oligopeptide portion encoding nucleic acid from the 5' end; The primer R2 includes another enzyme recognition site, a stop codon and a reverse complementary sequence of another part of the nucleic acid encoding the oligopeptide from the 5' end; B2) using the template 1 and the template 2 as templates, and using the primer F1 and the primer R1 to perform a first round of PCR amplification to obtain a first round of amplification products; B3) using the first round amplification product as a template, performing a second round of PCR amplification with the primers F2 and R2 to obtain a second round amplification product with an introduced enzyme recognition site, and then recovering a product of the second round amplification product that matches the size of the nucleic acid encoding the composite peptide to obtain the nucleic acid encoding the composite peptide; B4) constructing the encoding nucleic acid of the composite peptide into a prokaryotic expression vector through the introduced restriction site to obtain a recombinant plasmid; then transferring the recombinant plasmid into Escherichia coli to obtain a recombinant bacterium; inducing and culturing the recombinant bacterium with IPTG, collecting the induced culture product, and obtaining a composite peptide; the composite peptide includes a plurality of oligopeptides repeatedly connected in series.

4. The method according to claim 3, characterized in that: In the primer F2, a nucleic acid sequence of a tag for purification is also included between the enzyme recognition site and the oligopeptide portion encoding nucleic acid; In the primer R2, the reverse complementary sequence of the nucleic acid sequence of the tag for purification is also included between the stop codon and the reverse complementary sequence of the nucleic acid encoding another part of the oligopeptide.

5. The method according to claim 3 or 4, characterized in that: The oligopeptide is GnRH; And / or, the composite peptide is GnRHn, wherein n represents the number of GnRHs connected in series.

6. The method according to any one of claims 3 to 5, characterized in that: In step B4), the IPTG induction culture conditions are 0.01-1.5 mmol / L IPTG, 25-37° C. to induce expression for 6-8 hours; Or, in step B4), the IPTG induction culture conditions are 0.3 mmol / L IPTG, and induced expression at 37° C. for 8 h.

7. The method according to any one of claims 3 to 6, characterized in that: Step B4) further includes the steps of ultrasonically crushing and purifying the induced culture product to obtain the target composite peptide; Or the composite peptide is GnRH30.

8. The method according to any one of claims 1 to 7, characterized in that: The nucleotide sequence of the template 1 is sequence 1; Or, the nucleotide sequence of template 2 is sequence 2; Or, the nucleotide sequence of the primer F1 is sequence 3; Or, the nucleotide sequence of the primer R1 is sequence 4; Or, the nucleotide sequence of the primer F2 is sequence 5; Alternatively, the nucleotide sequence of the primer R2 is sequence 6.

9. The method according to any one of claims 3 to 8, characterized in that: The enzymes required for the first round of PCR amplification and the second round of PCR amplification are both Pfu enzyme and Ex enzyme.

10. Use of the method according to claim 1 or 2, the amplification template and the primers according to claim 1 or 2 in PCR amplification of multiple repeated short fragments or sequences in series; Or preparing a multiple oligopeptide encoding nucleic acid or a repeated tandem oligopeptide encoding nucleic acid by the method of claim 1 or 2; Or the method according to any one of claims 3 to 9, the use of the amplification template and the primers according to any one of claims 3 to 9 in preparing a composite peptide obtained by connecting multiple oligopeptides in series; or a method for preparing a nucleic acid encoding a composite peptide obtained by serially connecting multiple oligopeptides, comprising steps B1-B3 of any one of the methods of claims 3-9; Or a nucleic acid encoding a composite peptide prepared by any one of the methods of claims 3-9; Or a composite peptide prepared by the method according to any one of claims 3-9.

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