CN112062858A - Tandem protein for diagnosing alveolar echinococcosis and cloning expression method thereof - Google Patents

Abstract

The invention discloses a tandem protein for diagnosing alveolar echinococcosis and a cloning expression method thereof, the tandem protein is formed by connecting EmAgB3 gene and Em18 gene in series through GGGS, converting E.coli and carrying out induced expression, and the tandem protein has much larger molecular weight than the original protein and enhanced antigenicity. This lays the foundation for establishing more sensitive and specific serological diagnosis method, vaccine development and immunoprophylaxis.

Description

Tandem protein for diagnosing alveolar echinococcosis and cloning expression method thereof

Technical Field

The invention relates to the field of genetic engineering and proteomics, in particular to a tandem protein for diagnosing alveolar echinococcosis and a cloning expression method thereof.

Background

Echinococcosis vesiculosa (AE) is a zoonotic parasitic worm disease caused by infection of the tapetum larvae of Echinococcus multilocularis (Em). The disease is prevalent in parts of the countries of the middle, japan, russia, central asia and north america. Radical hepatectomy is the only reliable treatment method at present. Therefore, early immunodiagnosis of AE is a key requirement to reduce mortality of the disease. Early diagnosis of AE and taking timely measures can significantly reduce the low mortality rate caused by AE. At present, the diagnosis of the disease, especially the confirmation of the disease, also depends mainly on the etiology and imaging diagnosis. However, imaging diagnosis requires imaging background knowledge, is complicated to operate, and is not easy to identify smaller and atypical lesions, particularly, is not easy to distinguish from abscesses or tumors, and currently, a diagnostic method based on enzyme-linked immunosorbent assay (Em 2-ELISA) of the purified natural antigen Em2 has been recommended by the world health organization for serodiagnosis of AE.

Disclosure of Invention

The invention aims to provide a tandem protein for diagnosing echinococcosis vesiculosus.

The tandem protein provided by the invention isEscherichia coli pET28a-BL21(DE3)/01/EmAgB3-GGGS-Em18 (hereinafter abbreviated as EmAgB3-GGGS-Em18), the protein is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019622 and the preservation date of 2019, 8 months and 12 days.

Em18 is a novel protein epitope of AE, has remarkable antibody reactivity,Em18 is readily detected by immunoblotting and is a good serological marker tag for identification of other parasitic diseases, as well as active and inactive AE. The isotype of EmAgB3 antigen is directed against the specific immunoreactivity of the serum of patients with active AE, the gene is cloned and prokaryotic expression of bacteria is carried out (rEmAgB 3), and immunoblotting shows that the sensitivity and specificity of the protein are respectively 90.9% (80/88) and 98.5% (597/606), which shows that EmAgB3 can be applicable to serodiagnosis and follow-up monitoring of AE. And the use of the rmeagb 3 is simple and highly reproducible, and may be suitable for clinical diagnosis of AE patients and large-scale epidemiological investigation of epidemic areas. In view of the good immunogenicity of the EmAgB3 and Em18 proteins, and in order to increase the antigenicity of the proteins, the proteins were expressed in tandem using a linker protein GGGS, in order to increase the detection rate of AE.

The invention also provides a clone expression method of tandem protein EmAgB3-GGGS-Em18, which comprises the following steps:

(1) amplification of the EmAgB3 and Em18 genes:

amplifying an EmAgB3 gene, wherein an upstream primer introduces a BamHI restriction site, a downstream primer introduces a HindIII restriction site, and the primer sequences are as follows:

EmAgB3F：5´-CGGATCCGATGATGATGAAGTGACC-3´；

EmAgB3R：5´-CAAGCTTCTACTCATCCTCTTTAAT-3´；

amplifying the Em18 gene, wherein BamHI enzyme cutting sites are introduced into upstream and downstream primers, and the sequences of the primers are as follows:

Em18F： 5´-GGATCCGAAGGAGTCTGACTTAGCGGAT-3´；

Em18R：5´-GGATCCTATTTGAGGTTGGCCAGCTTCGT-3´。

(2) gene synthesis of EmAgB3-GGGS-Em 18:

biosynthesis was carried out based on the gene sequences of EmAgB3 and Em18, with NdeI cleavage sites added upstream and XhoI cleavage sites added downstream, and the linker GGGS protein was added between EmAgB3 and Em 18.

(3) And (3) carrying out enzyme digestion on the vector:

carrying out double enzyme digestion on the expression vector PET28a respectively, wherein the enzyme digestion system is as follows: pET28a 1. mu.g, 10. mu.L of FD Buffer 5. mu.L, NdeI 1. mu.L (10U/. mu.L), XhoI 1. mu.L (10U/. mu.L), ddH₂O 42μL；

(4) And (3) connecting the target fragment with a vector:

and respectively connecting the recovered and purified EmAgB3-GGGS-Em18 DNA fragments with a linearized pET28a vector, wherein a connector system is as follows: 2 × Seamless cloning Master Mix 10 μ L, Linear pGEX-6P-1/pET28a 3 μ L, EmAgB3/EmAgB3-GGGS-Em 185 μ L, Sterilized ddH₂O 2μL；

(5) Coli DH5 α competent cells were transformed and clones were screened:

transforming the connecting liquid into E.coli DH5 alpha competent cells, detecting, screening out positive clones, sequencing and identifying, propagating and extracting a recombinant pET28a plasmid vector;

(6) inducing expression of the fusion protein:

taking 1uL of pGEX-6P-1/pET28a vector after identifying and recombining to transform BL21(DE3), inoculating the cultured bacterial liquid into 2L of LB liquid culture medium containing antibiotics with corresponding types and concentrations according to the proportion of 1:100, culturing at 37 ℃ and 220 rpm, adding IPTG with the final concentration of 0.5 mM when the OD value reaches 0.6, inducing at 20 ℃ and 220 rpm overnight, and centrifugally collecting cell thalli;

(7) and (3) protein purification:

the thalli is ultrasonically crushed, and the recombinant EmAgB3-GGGS-Em18 is purified by nickel agarose affinity chromatography.

According to the technical scheme provided by the invention, the EmAgB3 protein and the Em18 protein are expressed in series by means of a linker protein GGGS through a biological engineering technology, so that a fusion protein EmAgB3-GGGS-Em18 is obtained. The concentration of the fusion protein EmAgB3-GGGS-Em18 cloned and purified by the method is 0.5 mg/mL. The molecular weight of the tandem protein is greatly increased compared with the original protein, and the antigenicity is also enhanced. This lays the foundation for establishing more sensitive and specific serological diagnosis method, vaccine development and immunoprophylaxis.

Preservation description:

culture name:Escherichia coli pET28a-BL21(DE3)/01/EmAgB3-GGGS-Em18

the preservation organization: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (China center for cell communication)

Address: wuhan university in Wuhan City of Hubei province of China

The preservation date is as follows: 8/12/2019

Registration number of the preservation center: CCTCC NO: M2019622.

Detailed Description

The present invention will be described in detail with reference to specific embodiments. The following examples are presented to assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any manner.

Em Qinghai voles isolates used in the following examples were isolated, identified and stored in the laboratory in the field.

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

Example 1

Cloning and sequencing of EmAgB3 and Em18 genes.

Total RNA of preserved miracidium is extracted by adopting an Easy Pure RNA Kit RNA extraction Kit, and genomic DNA of the miracidium is extracted by QIAamp DNA Mini Kit DNA and is used as a template for amplifying EmAgB3 genes and Em18 genes respectively.

(1) Amplification of EmAgB3 Gene

The EmAgB3 gene was amplified using EasyScript One-Step RT-PCR Supermix RT-PCR amplification kit. The reaction system is as follows: the PCR Reaction contained 2. mu.L of template RNA, 0.5. mu.L of each upstream and downstream primer (EmAgB 3F, EmAgB 3R) at a concentration of 10. mu.M, 10. mu.L of 2 Xone-Step Reaction Mix, 0.5. mu.L of Easy Script One-Step Enzyme Mix, and 6.5. mu.L of ddH2O in a total volume of 20. mu.L. The reaction conditions are as follows: reverse transcription is carried out at 45 ℃ for 30 min, pre-denaturation is carried out at 95 ℃ for 3 min, and then 45-cycle amplification (denaturation at 95 ℃ for 1 min, annealing at 60 ℃ for 45 s, and extension at 72 ℃ for 1 min) and re-extension at 72 ℃ for 5 min are carried out, and then the product is stored at 4 ℃. mu.L of the PCR product was subjected to agarose gel electrophoresis at a concentration of 1.5% to detect the amplified product.

(2) Amplification of Em18 Gene

The Em18 gene was amplified using the Trans Direct Animal Tissue PCR Kit. The PCR reaction was carried out in 20. mu.L total, containing 2. mu.L of template DNA, 0.5. mu.L each of the upstream and downstream primers (Em 18F, Em 8R) at a concentration of 10. mu.M, 10. mu.L of 2 XTRICT Supermix (+ dye), 9. mu.L of ddH 2O. The reaction conditions are respectively as follows: the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 1 min, annealing at 55 ℃ for 45 s, and extension at 72 ℃ for 1 min, for 35 cycles. Then, the mixture is extended for 10 min at 72 ℃ and stored at 4 ℃. 2. mu.L of the PCR product was subjected to 1.5% agarose gel electrophoresis to detect the amplified product.

(3) PCR product gel recovery and purification and T vector cloning sequencing

After the target band on the agarose Gel was cut, the PCR products of EmAgB3 and Em18 genes were recovered and purified using Gel Extraction Kit recovery Kit, and were linked to pGEM-T vector, respectively, transformed into E.coli DH 5. alpha. competent cells, and then plated and inoculated on LB agar plates containing Ampcolicin (100. mu.g/mL), IPTG (40. mu.g/mL), and X-gal (100. mu.g/mL). The sample is placed upright until no flowing liquid is seen on the surface, turned over and cultured in an incubator at 37 ℃ for 24 hours, and white colonies are selected. After colony PCR identification, the bacterial colony is subjected to enrichment culture by using LB liquid culture medium containing Amp Ampciillin (100 mu g/mL), and then the bacterial liquid is sent to Shanghai biological engineering technical service company for sequencing. And after the sequencing result is returned, performing online comparison on the PCR product sequence and a reference sequence in a database by using the Blast function of the NCBI website.

2 mu L of PCR products of EmAgB3 and Em18 are pipetted by a pipette, agarose gel electrophoresis with the concentration of 1.5% is carried out to detect the amplified products, and as a result of ultraviolet gel imaging, target bands are visible, and the sizes of the bands are 213 bp and 495 bp respectively.

After PCR product gel recovery and purification of EmAgB3 and Em18 and pGEM-T vector clone sequencing, the comparison shows that the PCR product gel completely conforms to the sequence reported in NCBI database, and the EmAgB3 and Em18 genes of Em Qinghai vole isolates have no base difference such as base deletion and substitution with the reference sequence.

Example 2

Gene synthesis of EmAgB3-GGGS-Em 18.

On the basis of clone sequencing and sequence comparison of EmAgB3 and Em18 genes, correct EmAgB3 and Em18 nucleotide sequences are determined, and in order to ensure the correctness of subsequent clone expression, a direct biosynthesis mode is adopted to synthesize a gene sequence to be expressed.

Biosynthesis was carried out based on the sequence of EmAgB3 gene (EmuJ _ 000381600) and Em18 gene (accession No.: AY 513264), which had an NdeI cleavage site at the upstream and an XhoI cleavage site at the downstream. And after a connexin GGGS protein is added between EmAgB3 and Em18 and enzyme cutting sites and GGGS protein are added, a sequence to be expressed is synthesized by adopting a biosynthesis method according to a verified DNA sequence to be expressed. Agarose gel electrophoresis of the synthetic gene NdeI-EmAgB3-GGGS-Em18-XhoI results in clear bands.

Example 3

And (3) carrying out enzyme digestion on the vector.

The expression vector PET28a was subjected to double digestion, and the 50. mu.L digestion system was as follows:

pET28a	1 μg
		10 × FD Buffer	5 μL
NdeI	1 μL (10 U/μL)
		XhoI	1 μL (10 U/μL)
ddH2O	42 μL
		total up to	50 μL

And (3) putting the PCR tube prepared with the system into a 37 ℃ constant-temperature water bath for reaction for 2 h, and recovering and purifying the linear vector fragment subjected to enzyme digestion by using glue for downstream seamless cloning.

Example 4

The fragment of interest is ligated to the vector.

According to the BBI seamless cloning kit operation manual, the recovered and purified EmAgB3-GGGS-Em18 DNA fragments are respectively connected with a linearized pET28a vector, and the connection system is as follows

2 × Seamless cloning Master Mix	10 μL
		Linear pGEX-6P-1/pET28a	3 μL
EmAgB3/EmAgB3-GGGS-Em18	5 μL
		Sterilized ddH2O	2 μL
Total up to	20 uL

And placing the PCR centrifuge tube containing the connection mixed solution in a 50 ℃ PCR instrument for connection reaction for 20 min. Immediately after the reaction was completed, the centrifuge tube was placed on ice.

Example 5

Coli DH5 α competent cells were transformed and clones were screened.

And (3) transforming the connecting liquid into E.coli DH5 alpha competent cells, detecting, screening and sequencing positive clones, and then expanding propagation and extracting a recombinant pET28a plasmid vector for transforming BL21(DE3) competent cells.

Example 6

Inducing expression of the fusion protein.

1. Transformation of

The recombinant pGEX-6P-1/pET28a vector identified at 1uL was transformed into BL21(DE3), heat-shocked at 42 ℃ for 90 s, allowed to stand on ice for 2 min, and then applied to LB solid plate medium containing antibiotics (recombinant pET28a clone was cultured in LB solid medium containing 30. mu.g/mL kanamycin), and cultured overnight at 37 ℃.

2. Selection of optimal Induction conditions for Pilot culture

(1) LB liquid medium containing kanamycin to a final concentration of 30. mu.g/mL was prepared separately for culturing the above BL21(DE3) bacterium transformed with the recombinant pET28a vector.

(2) A single colony of the expression strain BL21(DE3) of the above-described transformed recombinant vector was picked up and cultured overnight on a shaker at 37 ℃ and 220 rpm in a test tube containing 10 mL of LB liquid.

(3) The overnight-cultured bacterial suspension was inoculated into 10 mL of LB medium containing the corresponding antibiotic at a ratio of 1:100, and cultured at 37 ℃ and 220 rpm.

(4) When the OD reached 0.6, IPTG was added to a final concentration of 0.5 mM at 220 rpm, and the mixture was induced overnight at 20 ℃ and for 4 hours at 37 ℃ respectively, and a negative control was obtained without IPTG inducer.

(5) Centrifuging at 5000 rpm for 10 min to collect thallus, discarding supernatant, suspending thallus with 500 μ L PBS (pH7.4) buffer solution, ultrasonicating for 6 min, stopping for 1.5 s for 0.5 s, centrifuging respectively to collect supernatant and precipitate, dissolving the precipitate with 500 μ L inclusion body dissolving solution (8M Urea, 50 mM Tris-HCl, 300 mM NaCl, pH 8.0), mixing 40 μ L sample with 10 μ L5 × protein loading buffer, and boiling for 10 min.

(6) SDS-PAGE was performed using 5% concentrated gel and 12% separation gel. Loading 10 μ L of the sample, concentrating the gel for 80V 20 min, separating the gel for 120V 60 min, dyeing the gel with Coomassie brilliant blue R250 after electrophoresis, decolorizing and observing.

SDS-PAGE analysis of the small sample revealed a band of the desired size of the protein of interest at around 35 kDa.

3. Large scale inducible expression of fusion proteins

Inoculating the cultured bacterial liquid into 2L LB liquid culture medium containing antibiotics of corresponding species and concentration according to the proportion of 1:100, culturing at 37 ℃ and 220 rpm, adding 0.5 mM IPTG (isopropyl thiogalactoside) at the final concentration when the OD value reaches 0.6, inducing overnight at 20 ℃ and 220 rpm, and centrifugally collecting cell thallus.

Example 7

And (4) purifying the protein.

1. Ultrasonic crushing thallus

The collected bacterial cells were suspended in a disruption buffer (50 mM Tris, 300 mM NaCl, 0.2 mM PMSF, pH 8.0), and the cells were sonicated in an ice bath at 400W for 20 min (sonication for 2 s, pause for 6 s for one cycle). After the ultrasonic treatment is finished, the mixture is centrifuged at 12000 rpm and 4 ℃ for 30 min, and the supernatant is collected for further purification.

2. Nickel sepharose affinity chromatography of recombinant EmAgB3-GGGS-Em18

(1) Binding buffer (1 mM Tris, 300 mM NaCl, pH 8.0), Wash buffer (1 mM Tris, 300 mM NaCl, 20 mM Imidazole, pH 8.0) and Elution buffer (1 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH 8.0) were prepared.

(2) 5 mL of Ni-NTA was taken and the equilibration column was washed with Binding buffer 10 bed volumes at a flow rate of 5 mL/min.

(3) And (5) loading the mixture on a column, wherein the flow rate is 2 mL/min, and collecting filtrate.

(4) Binding buffer at 20 bed volumes washes the column with a flow rate of 5 mL/min.

(5) Washing impurities by a Wash buffer at the flow rate of 5 mL/min, and collecting the eluent.

(6) Eluting with Elution buffer at the flow rate of 2 mL/min, and collecting the eluate.

(7) The collected fractions were subjected to SDS-PAGE, and the 4 fractions having the highest purity were dialyzed against dialysate A (500 mM L-Arginine, 50 mM Tris, 300 mM NaCl, pH 8.0) for 16 hours, then against dialysate B (250 mM L-Arginine, 50 mM Tris, 300 mM NaCl, pH 8.0) for 4 hours, then against 1 XPBS having a pH of 7.4 for 6 hours, and then subjected to ultrafiltration concentration, centrifugation, filtration, and storage at-80 ℃ in 1 mL/tube.

The EmAgB3-GGGS-Em18 fusion protein is identified by SDS-PAGE after being respectively purified by nickel agarose affinity chromatography, and a target protein band with an expected size appears near 35 kDa.

Example 8

And (5) detecting the purified protein.

1. SDS-PAGE detection

SDS-PAGE was performed using 5% concentrated gel and 12% separation gel. Loading 1 μ g of the gel, concentrating the gel for 80V 20 min, separating the gel for 120V 60 min, dyeing the gel with Coomassie brilliant blue R250 after electrophoresis, decolorizing and observing.

The target protein is purified, and SDS-PAGE electrophoresis analysis shows that an obvious band appears at a corresponding position, which indicates that the fusion protein is successfully expressed and purified.

Western blot detection

(1) Preparing glue: preparing polyacrylamide gel, wherein the concentration gel is 5 percent, and the separation gel is 12 percent.

(2) Preparing a sample: the loading amount is 1 ug.

(3) Electrophoresis: performing gel concentration 80V electrophoresis for 30 min, and separating gel 120V electrophoresis for 60 min.

(4) Film transfer: and (5) carrying out 250 mA wet transfer for 90 min.

(5) And (3) sealing: 5% skimmed milk powder, slowly shaking at 37 deg.C and sealing for 2 hr.

(6) Primary antibody incubation: 1:500 diluted primary antibody of rabbit anti-his tag, incubated for 60 min at 37 ℃ with slow shaking in a constant temperature shaker.

(7) And (3) secondary antibody incubation: 1, diluting goat anti-rabbit secondary antibody at 8000, and incubating for 60 min at 37 ℃ with slow oscillation.

(8) Color development: TMB color development.

Example 9

Protein concentration determination: the final protein concentration is determined by using an SK3071 non-interference protein quantitative kit, and the volume of the sample to be detected is 20 muL. As a result, the protein concentration of EmAgB3-GGGS-Em18 was 0.5 mg/mL.

Sequence listing

<110> academy of veterinary sciences of animal husbandry in Qinghai province

QINGHAI PROVINCIAL PEOPLE'S Hospital

<120> tandem protein for diagnosing alveolar echinococcosis and cloning expression method thereof

<130> 1

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 227

<212> PRT

<213> Escherichia coli pET28a-BL21(DE3)/01/EmAgB3-GGGS-Em18

<400> 1

Asp Asp Asp Glu Val Thr Gln Thr Lys Lys Gly Val Met Lys Ala Ile

1 5 10 15

Ser Glu Ile Lys His Phe Phe Gln Ser Asp Pro Leu Gly Lys Lys Leu

20 25 30

Val Glu Val Met Lys Glu Val Gly Ser Val Cys Gln Met Val Arg Lys

35 40 45

Lys Ala Arg Met Ala Leu Lys Glu Tyr Val Arg Lys Leu Ile Lys Glu

50 55 60

Asp Glu Gly Gly Gly Ser Glu Ser Asp Leu Ala Asp Met Lys Asn Lys

65 70 75 80

Ala Ser Ala Tyr Glu Ser Lys Ile Ala Glu Leu Glu Met Leu Leu Gln

85 90 95

Gln Glu Arg His Ala Arg Glu Ser Leu Gln Lys Ser Gln Asp Lys Leu

100 105 110

Ala Glu Met Asn Arg Lys Leu Lys Glu Glu Thr Ala Ala Ser Ala Glu

115 120 125

Arg Asp Arg Leu Met Ala Gln Arg Asp Glu Val Gln Arg Glu Val Glu

130 135 140

Ala Gln Lys Val Ala Met Ala Lys Lys Glu Ala Glu Lys Ala Gln Ala

145 150 155 160

Glu Ala Glu Leu Arg Arg Met Arg Glu Lys His Asp Ala Lys His Lys

165 170 175

Ser Gln Val Asn Gly Ser Gly Asp Ala Ala Ser Gln Asp Asp Glu Ser

180 185 190

Glu Ala Lys Glu Leu Glu Val Ile Pro Asn Val Arg Arg Thr Glu Glu

195 200 205

Ser Arg Val Thr Ala Val Ser Lys Asn Glu Thr Leu Gln Thr Lys Leu

210 215 220

Ala Asn Leu

225

<210> 2

<211> 705

<212> DNA

<213> EmAgB3-GGGS-Em18 tandem protein (Escherichia coli pET28a-BL21(DE3)/01/EmAgB3-GGGS-Em18)

<400> 2

catatggatg atgatgaagt gacccagacg aagaagggtg tgatgaaggc cattagcgag 60

atcaagcact tcttccaaag tgatccattg ggcaaaaagt tggttgaagt aatgaaggaa 120

gtggggagtg tgtgccagat ggtgaggaag aaggcacgca tggcactgaa ggagtatgtc 180

agaaagttga ttaaagagga tgagtaggga ggaagcgagt ctgacttagc ggatatgaag 240

aataaggcat ctgcctatga gagtaagatt gcggagctgg agatgctgct acagcaggag 300

cgacatgcgc gtgagagtct tcagaagagc caagacaaac tggcggagat gaacagaaag 360

ctgaaggagg agactgcggc atcagccgaa gagcgcgacc gtctgatggc ccagcgtgac 420

gaagtgcaac gcgaagttga ggctcagaag gtcgccatgg ccaagaagga agctgaaaag 480

gctcaggctg aagctgagct tcgcagaatg cgtgagaaac acgatgcaaa gcacaagtcc 540

caggtcaatg gcagtggtga cgctgcttcg caggatgatg aaagtgaagc caaggaactt 600

gaggtgatac caaatgtgag gcggacggag gaatcgaggg tgacggccgt ctctaagaat 660

gagacgctcc agacgaagct ggccaacctc taactcgagc tcgag 705

Claims (3)

1. The tandem protein EmAgB3-GGGS-Em18 is characterized in that the tandem protein is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019622.

2. The tandem protein EmAgB3-GGGS-Em18 according to claim 1, wherein said tandem protein is used for the diagnosis of Echinococcosis vesiculosa.

3. A method for the clonal expression of the tandem protein emigb 3-GGGS-Em18 according to claim 1 or 2, characterized in that it comprises the following steps:

(1) amplification of the EmAgB3 and Em18 genes:

amplifying an EmAgB3 gene, wherein an upstream primer introduces a BamHI restriction site, a downstream primer introduces a HindIII restriction site, and the primer sequences are as follows:

EmAgB3F：5´-CGGATCCGATGATGATGAAGTGACC-3´；

EmAgB3R：5´-CAAGCTTCTACTCATCCTCTTTAAT-3´；

amplifying the Em18 gene, wherein BamHI enzyme cutting sites are introduced into upstream and downstream primers, and the sequences of the primers are as follows:

Em18F：5´-GGATCCGAAGGAGTCTGACTTAGCGGAT-3´；

Em18R：5´-GGATCCTATTTGAGGTTGGCCAGCTTCGT-3´；

(2) gene synthesis of EmAgB3-GGGS-Em 18:

according to gene sequences of EmAgB3 and Em18, NdeI enzyme cutting sites are added at the upstream, XhoI enzyme cutting sites are added at the downstream for biosynthesis, and a connexin GGGS protein is added between EmAgB3 and Em 18;

(3) and (3) carrying out enzyme digestion on the vector:

carrying out double enzyme digestion on the expression vector PET28a respectively, wherein the enzyme digestion system is as follows: pET28a 1. mu.g, 10. mu.L of FD Buffer 5. mu.L, NdeI 1. mu.L (10U/. mu.L), XhoI 1. mu.L (10U/. mu.L), ddH₂O 42μL；

(4) And (3) connecting the target fragment with a vector:

and respectively connecting the recovered and purified EmAgB3-GGGS-Em18 DNA fragments with a linearized pET28a vector, wherein a connector system is as follows: 2 × Seamless cloning Master Mix 10 μ L, Linear pGEX-6P-1/pET28a 3 μ L, EmAgB3/EmAgB3-GGGS-Em18 5μL，Sterilized ddH₂O 2μL；

(5) Coli DH5 α competent cells were transformed and clones were screened:

transforming the connecting liquid into E.coli DH5 alpha competent cells, detecting, screening out positive clones, sequencing and identifying, propagating and extracting a recombinant pET28a plasmid vector;

(6) inducing expression of the fusion protein:

taking 1uL of pGEX-6P-1/pET28a vector after identifying and recombining to transform BL21(DE3), inoculating the cultured bacterial liquid into 2L of LB liquid culture medium containing antibiotics with corresponding types and concentrations according to the proportion of 1:100, culturing at 37 ℃ and 220 rpm, adding IPTG with the final concentration of 0.5 mM when the OD value reaches 0.6, inducing at 20 ℃ and 220 rpm overnight, and centrifugally collecting cell thalli;

(7) and (3) protein purification:

the thalli is ultrasonically crushed, and the recombinant EmAgB3-GGGS-Em18 is purified by nickel agarose affinity chromatography.