CN107236027B

CN107236027B - Echinococcus granulosus TSP1 recombinant protein and soluble expression method and purification method thereof

Info

Publication number: CN107236027B
Application number: CN201710393428.0A
Authority: CN
Inventors: 景涛; 辛奇; 孙旭东; 袁苗苗; 李焕平; 高海军; 宋晓霞; 鲁俊; 那斌; 吕薇
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2017-05-27
Filing date: 2017-05-27
Publication date: 2020-10-16
Anticipated expiration: 2037-05-27
Also published as: CN107236027A

Abstract

The invention provides an echinococcus granulosus TSP1 recombinant protein, wherein the amino acid sequence of the echinococcus granulosus TSP1 recombinant protein is shown in a sequence table SEQ ID No. 2. The invention also provides a soluble expression method, which realizes the high-efficiency soluble expression of the TSP1 recombinant protein: most of the expressed recombinant protein is soluble protein, which accounts for 90% of the total soluble protein of the escherichia coli; the recombinant Echinococcus granulosus TSP1 was then purified using Ni-NTA His-bind (Clontech) pre-packed column to give TSP1 purified protein. A high purity Echinococcus granulosus TSP1 was obtained when elution buffer (500mM/L imidazole, 20mM/L Tris, 500mM/L NaCI, pH8.0) was used. The purified protein is used as a coating antigen, and an ELISA kit for detecting echinococcosis granulosa can be prepared.

Description

Echinococcus granulosus TSP1 recombinant protein and soluble expression method and purification method thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to echinococcus granulosus TSP1 recombinant protein, and a soluble expression method and a purification method thereof.

Background

Transmembrane protein 1(TSP1) is an antigenic gene in Echinococcus granulosus imago cDNA library. TSP1 is an important echinococcus granulosus antigen, may participate in echinococcus granulosus signaling pathway, and has important physiological significance for cell growth and differentiation. The TSP1 is possibly homologous gene with Annexin family (Annexin family) and belongs to Annexin family. The physiological function of the antigen is researched, so that the important significance of the antigen in life activities such as parasitism, growth and development of echinococcosis granulosa can be known, and a new drug target and a candidate vaccine can be provided for treating and preventing echinococcosis. Based on the research background, the invention constructs the prokaryotic expression plasmid for coding the Echinococcus granulosus TSP1 gene, converts the prokaryotic expression plasmid into an escherichia coli expression system, induces the soluble expression of the plasmid, and carries out protein purification to obtain the protein, thereby laying a foundation for the immunogenicity, biochemical characteristic analysis and functional research in the future.

Disclosure of Invention

The invention provides a prokaryotic expression vector pET30a-TSP1 of Echinococcus granulosus TSP1 by a genetic engineering technology, and the vector is used for transforming escherichia coli (BL21-DE3) to realize high-level soluble expression of TSP 1. And provides an affinity chromatography purification method, purifies a large amount of high-purity recombinant TSP1, and is used for analyzing immunogenicity and biochemical characteristics of TSP1, researching functions, developing anti-hydatid vaccines and anti-hydatid drugs and carrying out immunodiagnosis on cystic echinococcosis patients.

The invention provides an echinococcus granulosus TSP1 recombinant protein, wherein the amino acid sequence of the echinococcus granulosus TSP1 recombinant protein is shown in a sequence table SEQ ID No. 2.

Preferably, the nucleotide sequence coded by the echinococcus granulosus TSP1 recombinant protein is the base from position 27 to position 836 in the sequence table SEQ ID No. 1.

The invention also provides soluble expression of the echinococcus granulosus TSP1 recombinant protein, which comprises the following steps:

(1) amplifying a target gene TSP 1;

(2) constructing TSP1 expression plasmid: after the target gene TSP1 prepared in the step (1) is subjected to enzyme digestion and purification, a corresponding polyclonal enzyme cutting site of pET-30a is constructed, and a plasmid pET30a-TSP1 is constructed;

(3) transforming the plasmid pET30a-TSP1 prepared in the step (2)E.ColiBL21(DE3) competent cellsE.ColiBL21(DE3) -pET30a-TSP1 is subjected to scale-up culture, and then 0.2 mmol/L IPTG is added to induce shaking culture at 25 ℃ and 120 r/min for 8 hours; after centrifugation and resuspension, the supernatant was collected by sonication.

Preferably, the expanding culture in step (3) is carried out byE.ColiBL21(DE3) -pET30a-TSP1 was inoculated on LB solid medium and incubated overnight at 37 ℃; then, selecting a single clone on the culture medium to be put into an LB liquid culture medium, and carrying out shaking culture at 37 ℃ and 180r/min for 3 hours to enable the OD value to be 0.5; and finally, inoculating the bacterial liquid into an LB liquid culture medium, and performing shaking culture at 37 ℃ and 180r/min to enable the OD value to be 0.5.

The invention also provides a purification method of the echinococcus granulosus TSP1 recombinant protein, which is to purify the recombinant protein by using a Ni-NTA His-bind (Clontech) purification system and then elute by using an elution buffer solution; the formula of the elution buffer solution is as follows: 500mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH 8.0.

Preferably, the steps are as follows:

(1) adding ultrapure water into the Ni-NTA His-Bind prepacked column, and enabling the ultrapure water to slowly flow through a medium filled in the column;

(2) adding a binding buffer; the formula of the binding buffer solution is as follows: 20mM/L Tris, 500mM/L NaCl, 20mM/L imidazole, pH 8.0;

(3) adding supernatant extracted by ultrasonic disruption E.Coli BL21(DE3) -TSP1, standing, and slowly flowing the supernatant through an in-column medium;

(4) adding a binding buffer solution, and enabling the binding buffer solution to slowly flow through a medium in the column; the formula of the binding buffer solution is as follows: 20mM/LTris, 500mM/L NaCl, 20mM/L imidazole, pH 8.0;

(5) adding No.1 eluent to enable the No.1 eluent to slowly flow through a medium in the column, wherein the formula of the No.1 eluent is as follows: 100mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH 8.0;

(6) adding No.2 eluent to enable the No.2 eluent to slowly flow through a medium in the column, wherein the formula of the No.2 eluent is as follows: 200mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0;

(7) adding No. 3 eluent to enable the No. 3 eluent to slowly flow through a medium in the column, wherein the formula of the No. 3 eluent is as follows: 300mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0;

(8) adding No. 4 eluent to enable the No. 4 eluent to slowly flow through a medium in the column, wherein the formula of the No. 4 eluent is as follows: 400mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH 8.0;

(9) adding No. 5 elution buffer solution, enabling the buffer solution to slowly flow through a medium in the column, and collecting eluent; the formula of the No. 5 elution buffer solution is as follows: 500mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH 8.0.

Preferably, the volume ratio of the supernatant extracted by the ultrasonication of E.coli BL21(DE3) -TSP1 to the elution buffer is 1: 1.

The invention also provides application of the echinococcus granulosus TSP1 recombinant protein in preparation of an ELISA kit for detecting echinococcosis granulosis.

The invention also provides an ELISA kit for detecting echinococcosis granulosus, and the coating antigen in the ELISA kit is the echinococcus granulosus TSP1 recombinant protein.

Preferably, the coating antigen is diluted with 0.05M/L carbonate buffer pH 9.6 to a final concentration of 8. mu.g/ml;

preferably, the kit further comprises a blocking solution, wherein the blocking solution is TBST containing 5% skimmed milk powder, and the unit of the percentage is g/ml.

The invention provides a novel echinococcus granulosus transmembrane protein 1(TSP1) gene and a protein coded by an echinococcus granulosus TSP1 gene, and provides a prokaryotic expression vector pET30a-TSP1 of echinococcus granulosus TSP 1. The vector contains a T7 promoter, a terminator, a bacterial ribosome binding site, a TSP1 gene and a histidine tag, the TSP1 gene is cloned from an Echinococcus granulosus imago cDNA library, the T7 promoter is used for controlling the expression of the gene in Escherichia coli, the vector is used for transforming Escherichia coli (BL21-DE3), and the bacterium is induced under the conditions of lower temperature, lower inducer concentration and shorter induction time, so that the efficient soluble expression of TSP1 can be realized: most of the expressed recombinant protein is soluble protein, and accounts for 90% of the total soluble protein of the escherichia coli. Then, the recombinant echinococcus granulosus TSP1 was purified by Ni-NTA superflow affinity chromatography to obtain TSP1 purified protein. A novel high purity of the granulosa echinococcus TSP1 was obtained using 10ml of elution buffer (500mM/L imidazole, 20mM/L Tris, 500mM/LNaCI, pH 8.0). The TSP1 recombinant protein has high expression amount and is soluble expressed under specific induction conditions, the operation for purifying the protein is quite simple, the cost is low, and the protein is extremely easy to reuse. The recombinant protein prepared by the invention can be used for immunodiagnosis of cystic echinococcosis patients, can be identified by serum of cystic echinococcosis patients as an immune antigen, has higher specificity and sensitivity when being applied to indirect ELISA detection, and has a clinical detection coincidence rate as high as 90%.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows the result of electrophoresis of PCR amplification products of Echinococcus granulosus TSP1 gene;

FIG. 2 is a prokaryotic expression of Echinococcus granulosus TSP 1;

FIG. 3 shows the result of affinity chromatography purification of Echinococcus granulosus TSP 1.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.

Example an Echinococcus granulosus TSP1 Gene sequence and the protein sequence encoded by it

The total length of Echinococcus granulosus TSP1 is 1047 nucleotides, the largest Open Reading Frame (ORF) is located at positions 27-836, and contains 810bp, the initiation codon is atg, the termination codon is tga, and 269 amino acids are coded, and the nucleotide sequence and the coded amino acid sequence are respectively shown in sequence tables SEQ ID No.1 and 2.

The nucleotide sequence is as follows:

amino acid sequence:

example cloning of the Echinococcus bifineus TSP1 prokaryotic expression vector pET30a-TSP1

1. Amplification of target Gene TSP1

Using Echinococcus granulosus pBluescript II SK-TSP1 (Echinococcus granulosus pBluescript II SK-TSP1 available from the institute of medicine, Hainan institute of medicine, and from the institute of society, the following two primer pairs were designed based on the sequence of Echinococcus granulosus TSP1 gene:

the sequence of the upstream primer is as follows: CGCGATATCATGACCACTGG, containingEcoRV, enzyme cutting sites of the protease,

the sequence of the downstream primer is as follows: GAGTCGACTCATATTGGGGAAGC, containingSalI, enzyme cutting sites of the protease,

amplifying a gene sequence (namely a 27-836 gene sequence) in the maximum Open Reading Frame (ORF) of the TSP1 gene, and carrying out PCR (polymerase chain reaction) under the following conditions: pre-denaturation at 98 ℃ for 5 min; denaturation at 96 ℃ for 35s, renaturation at 65 ℃ for 35s, extension at 72 ℃ for 45s, and 35 cycles; extension at 72 ℃ for 10 min. The electrophoresis results of the amplification products are shown in FIG. 1.

FIG. 1 shows the result of electrophoresis of PCR amplification products of Echinococcus granulosus TSP1 gene; wherein, M, Marker III; 1, TSP1PCR amplification product.

2. Construction of TSP1 expression plasmid

After the PCR product is purified, the amplified fragment is obtainedEcoRV andSali, after enzyme digestion and purification, constructing a corresponding polyclonal enzyme cutting site of pET-30a, constructing a plasmid pET30a-TSP1, and identifying the sequence information and the target gene sequence of TSP1 through sequencingColumn identity: the nucleotide sequence is 27 th to 836 th positions in the sequence table SEQ ID No.1, 269 amino acids are coded, and the coded amino acid sequence is the sequence table SEQ ID No. 2.

Example prokaryotic expression of Echinococcus triphinatus TSP1

Under different induction conditions, the soluble expression amount of the recombinant protein is different, and the optimal induction method is as follows:

preparation ofE.ColiBL21(DE3) competent cells, into which the plasmid pET30a-TSP1 prepared in example 2 was transformedE.ColiIn BL21(DE3) competent cells, streaking was performedE.ColiBL21(DE3) -pET30a-TSP1 was incubated overnight at 37 ℃ on LB solid medium; selecting a single clone on the culture medium to be placed into 5ml LB liquid culture medium, and carrying out shaking culture for 3 hours at 37 ℃ and 180r/min to ensure that the OD value is 0.5; inoculating 1ml of bacterial liquid into 200ml of LB liquid culture medium, and carrying out shaking culture at 37 ℃ and 180r/min until the OD value is 0.5; adding IPTG (0.2 mmol/L) to induce shaking culture at 25 deg.C and 120 r/min for 8 hr; centrifugation at 12000g/min for 10min at 4 ℃ collected bacteria, resuspended in 40ml binding buffer (20 mM/L Tris, 500mM/L NaCl, 20mM/L imidazole, pH 8.0); the bacteria are broken by ultrasonic, the power is 200W, the ultrasonic is 8S, the interval is 8S, the supernatant is extracted after 25mins, and SDS-PAGE electrophoresis is carried out, so that the expression level of the protein in the supernatant is very high and the protein is expressed in a soluble way (see figure 2). The majority of the recombinant protein is soluble protein, which accounts for 90% of the total soluble protein of the Escherichia coli.

In order to find the optimal induction conditions, applicants conducted a number of experiments, and FIG. 2 shows prokaryotic expression of Echinococcus granulosus TSP1 under different induction conditions.

Wherein, in the A diagram, M: marker; 1-4, BL21(DE3) -pET30a-TSP1 at 37 ℃, IPTG concentrations of 1, 0.8, 0.5 and 0.2 mmol/L respectively, and an electrophoretogram of supernatant after 18 h of induction; 5-8, BL21(DE3) -pET30a-TSP1 at 30 ℃, IPTG concentration of 1, 0.8, 0.5 and 0.2 mmol/L respectively, and an electrophoretogram of supernatant after 12 h of induction;

in Panel B, M: marker; 1-3, BL21(DE3) -pET30a-TSP1 at 25 ℃ and IPTG of 0.5, 0.2 and 0.1 mmol/L respectively, and an electrophoretogram of supernatant after 8h of induction.

As can be seen from figure 2, TSP1 with extremely low expression level and soluble expression is visible in the supernatant after 18 h of induction at the induction temperature of 37 ℃ and IPTG of 1, 0.8, 0.5 and 0.2 mmol/L respectively; reducing the induction temperature to 30 ℃, respectively using IPTG (isopropyl-beta-thiogalactoside) of 1, 0.8, 0.5 and 0.2 mmol/L, inducing for 12 hours, and obtaining soluble TSP1 in the supernatant, wherein the expression amount is increased compared with that at 37 ℃; the induction temperature is further reduced to 25 ℃, the IPTG concentrations are respectively 0.5, 0.2 and 0.1 mmol/L, and after 8 hours of induction, the expression level of TSP1 in the supernatant can be increased, the IPTG concentration is 0.2 mmol/L, and the expression level of soluble TSP1 is the maximum, so the condition is used as the optimal condition for TSP1 prokaryotic induction expression.

Example affinity chromatography purification of Echinococcus tetrastiglium TSP1

The target protein was purified using a Ni-NTA His-bind (Clontech) purification system. Ni-NTA His-Bind prepacked column was purchased from Novagen, USA, model: 70691-3.

When each substance was dropped, the dropping speed was 1 drop/10 seconds.

The purification effect of the recombinant protein obtained by different purification methods is different, and the purification method needs to be optimized. The following is part of the optimization process:

adding 20ml of ultrapure water into the Ni-NTA His-bind prepacked column, and enabling the ultrapure water to slowly flow through a medium filled in the column; 20ml of binding buffer (50 mM/L NaH) was added₂PO₄300mM/L NaCl, pH8.0) to be slowly flowed through the medium filled in the column; loading, adding 4ml, and ultrasonic crushingE.ColiBL21(DE3) -pET30a-TSP1 extracting supernatant, controlling the flow rate to make the supernatant slowly flow through the medium in the column and collecting; 40ml of rinsing buffer (50 mM/L NaH) was added₂PO₄300mM/L NaCl, 20mM/L imidazole, pH8.0), the flow rate is controlled so that the mixed protein is slowly flowed through the medium in the column to elute the mixed protein; 50mM/L imidazole eluent (50 mM/L imidazole, 50mM/L NaH) was added₂PO₄300mM/L NaCl, pH8.0) 2ml, and allowing to slowly flow through the medium in the column and collect; adding 100mM/L imidazole eluent (100 mM/L imidazole, 50mM/L NaH)₂PO₄300mM/L NaCl, pH8.0) 2ml, and allowing to slowly flow through the medium in the column and collect;adding 200mM/L imidazole eluent (200 mM/L imidazole, 50mM/L NaH)₂PO₄300mM/L NaCl, pH8.0) 2ml, and allowing to slowly flow through the medium in the column and collect; adding 300mM/L imidazole eluent (300 mM/L imidazole, 50mM/L NaH)₂PO₄300mM/L NaCl, pH8.0) 2ml, and allowing to slowly flow through the medium in the column and collect; adding 400mM/L imidazole eluent (400 mM/L imidazole, 50mM/L NaH)₂PO₄300mM/LNaCl, pH8.0) 2ml, was allowed to flow slowly through the column media and collected. The collected eluates were subjected to SDS-PAGE analysis to examine the degree of protein purification, respectively, as shown in Panel A in FIG. 3.

Wherein, in diagram A, M: marker; 1-2, BL21(DE3) -pET30a-TSP1 supernatant as such; 3, after the protein is loaded on the column, flowing through the liquid; 4, 50mM/L imidazole eluent; 5,100 mM/L imidazole eluate; 6,200 mM/L imidazole eluate; 7,300 mM/L imidazole eluate; 8,400 mM/L imidazole eluent.

As can be seen from diagram a in fig. 3: the target protein can be eluted by respectively using imidazole eluents of 50mM/L, 100mM/L, 200mM/L, 300mM/L and 400mM/L, particularly the target protein amount eluted by the imidazole eluents of 200mM/L is the most, but the protein purity is not high, the impurity protein content is very high, so the protein purification conditions are further optimized, and the following method is used for purification.

Adding 20ml of ultrapure water into the Ni-NTA His-Bind pre-packed column, and enabling the ultrapure water to slowly flow through a medium filled in the column so as to flush the medium; add 20ml Tris-binding buffer (20 mM/L Tris, 500mM/L NaCl, 20mM/L imidazole, pH8.0) to equilibrate the media; loading, adding 10ml, and ultrasonic crushingE.ColiBL21(DE3) -TSP1, the flow rate is controlled to make the supernatant slowly flow through the column medium; adding 40ml Tris-binding buffer (20 mM/L Tris, 500mM/L NaCl, 20mM/L imidazole, pH8.0) and slowly flowing through the column medium to elute the hetero protein; 2ml of No.1 eluent (100 mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0) was added, and the mixture was allowed to slowly flow through the column medium and collected; adding an eluate No.2 (200 mM-L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0) 2ml, which was allowed to flow slowly through the column medium and collected; 2ml of eluent No. 3 (300 mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0) was added, and the mixture was allowed to slowly flow through the column medium and collected; 2ml of No. 4 eluent (400 mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0) was added, and the mixture was allowed to slowly flow through the column medium and collected; 2ml of No. 5 eluent (500mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0) was added, and the mixture was allowed to slowly flow through the column medium and collected; the collected eluates were analyzed by SDS-PAGE, respectively, to examine the degree of purification of the protein. It can be seen that TSP1 (see FIG. 3B) was obtained in very high purity when eluted with eluent No. 5, and the protein had a molecular weight of about 29.7 KD. The result of SDS-PAGE is shown in FIG. 3B.

In the B diagram of fig. 3, M: marker; 1-2, BL21(DE3) -pET30a-TSP1 supernatant as such; 3, after the protein is loaded on the column, flowing through the liquid; eluent No. 4 and No. 1; eluent No. 5 and No. 2; eluent No. 6 and No. 3; eluent No. 7 and 4; no. 8 and No. 5 eluent (good purification effect and high purity).

EXAMPLE five ELISA kits for detecting Echinococcosis granulosa

The ELISA kit for detecting echinococcosis granulosa comprises the following components:

1. coating antigen: diluting the purified recombinant protein obtained in example 4 as a coating antigen with 0.05M/L carbonate buffer solution of pH 9.6 to a final concentration of 8. mu.g/ml;

2. sealing liquid: 5g of skimmed milk powder is dissolved in 100ml of TBST;

3. negative control: diluting human serum at a ratio of 1: 200;

4. enzyme-labeled secondary antibody: diluting peroxidase-labeled goat anti-human IgG at a ratio of 1: 10000;

5. TMB color development liquid;

6. stopping liquid: 2mol/L concentrated sulfuric acid.

Example ELISA detection of Echinococcus hexakishinouye antigen TSP1

And (3) taking the purified recombinant protein as an antigen, and carrying out indirect ELISA detection on 20 parts of echinococcus infected patient serum samples and 20 parts of healthy human serum samples. The specific method comprises the following steps:

the purified recombinant protein was diluted to a final concentration of 8. mu.g/ml 200. mu.g/well with 0.05M/L carbonate buffer pH 9.6

Coating a 96-hole enzyme label plate, and standing overnight at 4 ℃; the wells were drained and the plates were washed 3 times (5 min/time) with PBST pH 7.4, then 200. mu.l of blocking solution was added to each well and blocked at 37 ℃ for 1 hour. PBST plate washing 3 times (5 min/time), 100 per well

Adding echinococcosis granulosa patient and healthy human serum (diluted 1: 200), respectively, and incubating at 37 deg.C for 1 hr; PBST washing plate 3 times, each hole 100 u l add peroxidase labeled sheep anti-human IgG (1: 10000 dilution), 37 degrees C temperature 1 h incubation; PBST washing plate 3 times, adding TMB color development solution into 100 μ L per well, reacting at 37 deg.C in dark place for 30 min, adding 2mol/L concentrated sulfuric acid into 50 μ L per well to terminate the reaction, and measuring absorbance ((

) The value is obtained. With serum of healthy persons

The mean value is 2 times plus the standard deviation to be a positive judgment value. The ELISA detection results are shown in Table 1, and the diagnostic sensitivity of the kit of the invention to the serum of echinococcosis granulosa patients is 90% (18/20), and the kit has better immunodiagnosis value for echinococcosis granulosa.

TABLE 1 Echinococcus granulosus TSP1 ELISA test results

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Lanzhou university

<120> Echinococcus granulosus TSP1 recombinant protein and soluble expression method and purification method thereof

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ctcaattctt gctaaaatac gatgttggaa taccggcgat attcatcgtg gtgggatttg 300

ccatcaccgg tgtctgcatt ctcgggttca tagcgacctg ctgttcatgc aataaactcc 360

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ctgatgcatt ggcgttgact ccaccggttg agggatgtcg tggaaagata agacgctacg 720

ctgatgaata catgttgaag attttggtca ttcttatcat cttcatcgct tgccaggtag 780

ttctttcaat cctcacctac gtggcactga catgtaagac tgcttcccca atatgagagg 840

aggggcgccg tttgctgcac caaattatct ctgcttgttt tctgcacctc atcattcact 900

actgtgttaa gagatgtggg ttcctctcat tgattcccct tcactcacaa ggaaagttaa 960

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Met Thr Thr Gly Tyr Asp Ile Met Arg Ser Ser Cys Ser Thr Leu Thr

1 5 10 15

Arg Phe Ile Ile Gly Leu Leu Asn Leu Ile Leu Gly Val Thr Phe Leu

20 25 30

Val Leu Gly Val Val Gly Val Leu Leu Arg Thr Asn Lys Glu Phe Leu

35 40 45

Leu Lys Phe Thr Asn Ser Leu Ser Glu Lys Phe Leu Asn Glu Ala Ser

50 55 60

Gln Glu Gln Ala Asp Glu Ile Ala Gln Phe Leu Leu Lys Tyr Asp Val

65 70 75 80

Gly Ile Pro Ala Ile Phe Ile Val Val Gly Phe Ala Ile Thr Gly Val

8590 95

Cys Ile Leu Gly Phe Ile Ala Thr Cys Cys Ser Cys Asn Lys Leu Leu

100 105 110

Gln Ile Tyr Ala Val Ile Leu Thr Ser Ile Val Val Ile Gln Val Ile

115 120 125

Ala Val Gly Val Ile Phe Gly Val Pro Asp Ile Tyr Arg Ser Leu Ala

130 135 140

Phe Gln Gly Met Glu Lys Thr Leu Ala Tyr Tyr Asp Pro Ser Thr Pro

145 150 155 160

Glu Gly Lys Gly Ser Ala Arg Leu Trp Asp Leu Ile Met Thr Ala Asn

165 170 175

Lys Glu Thr Cys Cys Gly Met Asp Gly Ala Asn Asp Phe Lys Asn Thr

180 185 190

Pro Phe Asp Pro Lys Cys Pro Lys Tyr Cys Cys Lys Ala Asp Lys Asp

195 200 205

Cys Leu Ile Thr Asp Ala Leu Ala Leu Thr Pro Pro Val Glu Gly Cys

210 215 220

Arg Gly Lys Ile Arg Arg Tyr Ala Asp Glu Tyr Met Leu Lys Ile Leu

225 230 235 240

Val Ile Leu Ile Ile Phe Ile Ala Cys Gln Val Val Leu Ser Ile Leu

245250 255

Thr Tyr Val Ala Leu Thr Cys Lys Thr Ala Ser Pro Ile

260 265

Claims

1. An echinococcus granulosus TSP1 recombinant protein suitable for immunodiagnosis of cystic echinococcosis patients, which is characterized in that: the amino acid sequence of the Echinococcus granulosus TSP1 recombinant protein is shown in SEQ ID No.2 of the sequence table.

2. The echinococcus granulosus TSP1 recombinant protein suitable for immunodiagnosis of cystic echinococcosis patients of claim 1, wherein: the nucleotide sequence of the gene for coding the Echinococcus granulosus TSP1 recombinant protein is shown as the 27 th-836 th base in the sequence table SEQID No. 1.

3. The method for the soluble expression of the echinococcus granulosus TSP1 recombinant protein suitable for immunodiagnosis of cystic echinococcosis patients of claim 1 or 2, wherein: the method comprises the following steps: (1) amplifying a target gene TSP1, wherein the nucleotide sequence of the target gene TSP1 is shown as 27 th-836 th bases in a sequence table SEQ ID No. 1; (2) constructing TSP1 expression plasmid: after the target gene TSP1 prepared in the step (1) is subjected to enzyme digestion and purification, a corresponding polyclonal enzyme cutting site of pET-30a is constructed, and a plasmid pET30a-TSP1 is constructed; (3) transforming the plasmid pET30a-TSP1 prepared in the step (2) into E.ColiBL21(DE3) competent cells, carrying out scale-up culture on the E.ColiBL21(DE3) -pET30a-TSP1, and then adding 0.2 mmol/L of IPTG to induce shaking culture at 25 ℃ for 8 hours; after centrifugation and resuspension, the supernatant was collected by sonication.

4. The method for the soluble expression of the echinococcus granulosus TSP1 recombinant protein suitable for immunodiagnosis in patients with cystic echinococcosis according to claim 3, wherein: the expansion culture in the step (3) is to inoculate E.ColiBL21(DE3) -pET30a-TSP1 on LB solid medium and incubate at 37 ℃ overnight; then, selecting a single clone on the culture medium to be put into an LB liquid culture medium, and carrying out shaking culture at 37 ℃ and 180r/min for 3 hours to enable the OD value to be 0.5; and finally, inoculating the bacterial liquid into an LB liquid culture medium, and performing shaking culture at 37 ℃ and 180r/min to enable the OD value to be 0.5.

5. The method for purifying the echinococcus granulosus TSP1 recombinant protein suitable for immunodiagnosis of cystic echinococcosis patients of claim 1 or 2, wherein: the recombinant protein was purified using a Ni-NTA His-Bind purification system and then eluted using an elution buffer.

6. The method for purifying the echinococcus granulosus TSP1 recombinant protein suitable for immunodiagnosis of cystic echinococcosis patients according to claim 5, wherein: the method comprises the following steps: (1) adding ultrapure water into the Ni-NTA His-Bind prepacked column, and enabling the ultrapure water to slowly flow through a medium filled in the column; (2) adding a binding buffer; the formula of the binding buffer solution is as follows: 20mM/LTris, 500mM/L NaCl, 20mM/L imidazole, pH 8.0; (3) adding supernatant extracted by ultrasonic crushing E.ColiBL21(DE3) -TSP1, standing, and slowly flowing the supernatant through a medium in a column; (4) adding a binding buffer solution, and enabling the binding buffer solution to slowly flow through a medium in the column; the formula of the binding buffer solution is as follows: 20mM/LTris, 500mM/L NaCl, 20mM/L imidazole, pH 8.0; (5) adding No.1 elution buffer solution, and enabling the elution buffer solution to slowly flow through a medium in the column, wherein the formula of the No.1 elution buffer solution is as follows: 100mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH 8.0; (6) adding No.2 elution buffer solution, and enabling the No.2 elution buffer solution to slowly flow through the medium in the column, wherein the formula of the No.2 elution buffer solution is as follows: 200mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0; (7) adding No. 3 elution buffer solution, and enabling the elution buffer solution to slowly flow through the medium in the column, wherein the formula of the No. 3 elution buffer solution is as follows: 300mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH8.0; (8) adding No. 4 elution buffer solution, and enabling the elution buffer solution to slowly flow through the medium in the column, wherein the formula of the No. 4 elution buffer solution is as follows: 400mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH 8.0; (9) adding No. 5 elution buffer solution, enabling the elution buffer solution to slowly flow through a medium in the column, and collecting the No. 5 elution buffer solution after the elution buffer solution passes through the column; the formula of the No. 5 elution buffer solution is as follows: 500mM/L imidazole, 20mM/L Tris, 500mM/L NaCl, pH 8.0.

7. The method for purifying the echinococcus granulosus TSP1 recombinant protein suitable for immunodiagnosis of cystic echinococcosis patients according to claim 6, wherein: the volume ratio of the supernatant extracted by the ultrasonic disruption of the E.ColiBL21(DE3) -TSP1 to the total amount of the elution buffer solution is 1: 1; the total amount of the elution buffer refers to the sum of the volumes of elution buffer No.1, elution buffer No.2, elution buffer No. 3, elution buffer No. 4 and elution buffer No. 5.

8. The use of the echinococcus granulosus TSP1 recombinant protein of claim 1 or 2 for immunodiagnosis of cystic echinococcosis patients in the preparation of an ELISA kit for detecting echinococcosis granulosis.

9. An ELISA kit for detecting echinococcosis granulosus, suitable for immunodiagnosis of cystic echinococcosis patients, characterized in that: the coating antigen in the ELISA kit is the Echinococcus granulosus TSP1 recombinant protein of claim 1 or 2.

10. The ELISA kit for detecting echinococcosis granulosa suitable for immunodiagnosis of patients with hydatid granulosa according to claim 9, wherein: the coating antigen was diluted with 0.05M/L carbonate buffer pH 9.6 to a final concentration of 8. mu.g/ml.

11. The ELISA kit for detecting echinococcosis granulosa suitable for immunodiagnosis of patients with hydatid granulosa according to claim 9, wherein: the kit also comprises a confining liquid, wherein the confining liquid is a solution containing 5g of skimmed milk powder per 100ml of TBST.