Prokaryotic expression and purification of Echinococcus multilocularis glutathione S-transferase, and preparation and application of polyclonal antibody

Abstract

The invention discloses prokaryotic expression and purification of Echinococcus multilocularis glutathione S-transferase, and preparation and application of a polyclonal antibody, and relates to the technical field of biology.The technical key point is that the preparation method of the polyclonal antibody of the Echinococcus multilocularis glutathione S-transferase comprises the following steps that S1, RNA in vesicle tissue of Echinococcus multilocularis is extracted, and cDNA is prepared; s2, carrying out PCR (Polymerase Chain Reaction) amplification by taking the cDNA as a template to obtain an EmGST gene which can encode an amino acid sequence as shown in SEQ ID NO: 1; s3, connecting the EmGST gene to a prokaryotic expression vector plasmid through a double digestion enzyme reaction to construct a recombinant vector, then transfecting the recombinant vector to competent escherichia coli, and inducing prokaryotic expression and purification to obtain a recombinant protein; and S4, immunizing a mouse to prepare antiserum by taking the echinococcosis multilocularis glutathione transferase as an antigen, and purifying the antiserum to obtain the polyclonal antibody of the echinococcosis multilocularis glutathione transferase. The ImGST protein polyclonal antibody prepared by the invention has the advantages of high titer and good specificity.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to prokaryotic expression and purification of glutathione S-transferase from Echinococcus multilocularis, preparation of polyclonal antibodies and application thereof. Background Art

[0002] Echinococcus multilocularis (Em) causes fatal alveolar echinococcosis. This disease is potentially fatal in animals and humans. Currently, treatment for alveolar echinococcosis usually includes surgery and antiparasitic drugs (such as albendazole). It is important to develop effective control strategies (including vaccination) to reduce the impact of this zoonosis on animals and humans.

[0003] GST is a group of multifunctional enzymes whose main functions include catalyzing the coupling of glutathione sulfate anions with various bisubstrates or transporting a series of hydrophobic ligands. GST is one of the key enzymes related to cellular detoxification and excretion of various physiological and foreign substances. Most worms contain active GST in their adult stages. GST is considered to be essential for the survival of helminth parasites. It can remove endogenous / exogenously derived toxic compounds and foreign organisms, such as reactive oxygen species, chemotherapeutic drugs and chemical drugs. GST helps parasites evade the host's immune response through its detoxification function and repair mechanism. However, there is still a lack of sufficient understanding as to whether EmGST has a role in regulating immune function and whether it is involved in the process of host immune regulation. In addition, the function of GST in Echinococcus multilocularis and whether it promotes the progression of alveolar echinococcosis have not been reported. Summary of the invention

[0004] In view of the above defects in the prior art, the purpose of the present invention is to provide a prokaryotic expression and purification of glutathione S-transferase from Echinococcus multilocularis, preparation of polyclonal antibodies and application thereof, by cloning prokaryotic expressed EmGST and introducing its in vitro activity into the RAW264.7 mouse macrophage inflammation model to investigate the inhibitory effect of EmGST on lipopolysaccharide (LPS)-induced macrophage inflammation, which helps to gain a deeper understanding of the inhibitory effect of EmGST on LPS-induced macrophage inflammation. The present invention also reveals the immunoregulatory mechanism of EmGST in the interaction between Echinococcus multilocularis and the host, providing a certain experimental basis for the study of EmGST to improve or prevent autoimmune diseases and allergies.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] The invention provides a glutathione S-transferase from Echinococcus multilocularis. The amino acid sequence of the glutathione S-transferase from Echinococcus multilocularis is shown in SEQ ID NO:1.

[0007] The present invention also provides a method for preparing the polyclonal antibody of the Echinococcus multilocularis glutathione S-transferase, comprising the following steps:

[0008] S1. obtaining the cystic tissue of Echinococcus multilocularis, extracting RNA from the cystic tissue of Echinococcus multilocularis, and preparing cDNA;

[0009] S2, using the cDNA in step S1 as a template, performing PCR amplification by reverse transcription polymerase chain reaction to obtain an EmGST gene encoding an amino acid sequence as shown in SEQ ID NO: 1;

[0010] S3, using upstream and downstream primers containing specific restriction sites to perform double enzyme reaction on the EmGST gene and plasmid, respectively, connecting the EmGST gene to the prokaryotic expression vector plasmid, constructing a recombinant vector, and then transfecting the recombinant vector into competent Escherichia coli cells, inducing prokaryotic expression and purification, and obtaining the recombinant protein;

[0011] S4. Using the amino acid sequence of Echinococcus multilocularis glutathione transferase as shown in SEQ ID NO: 1 as an antigen, immunizing mice to prepare antiserum, and purifying the antiserum to obtain polyclonal antibodies against Echinococcus multilocularis glutathione transferase.

[0012] Furthermore, in step S2, the primers used for the PCR amplification are: the forward primer is shown in SEQ ID NO: 2, and the reverse primer is shown in SEQ ID NO: 3.

[0013] Further, in step S2, the reaction system of the PCR amplification is: 2×Taq Master Mix 25 μL, 10 μm Forward Primer 2 μL, 10 μm Reverse Primer 2 μL, TEmplate cDNA <0.5 μg, ddH2O, Up to 50 μL;

[0014] The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 25-35 cycles, extension at 72°C for 30 s, and termination extension at 72°C for 2 min.

[0015] Furthermore, the upstream and downstream primers containing specific restriction sites are respectively an upstream primer containing an NdeI restriction site and a downstream primer containing a HindⅢ restriction site. The nucleotide sequence of the upstream primer containing the NdeI restriction site is shown in SEQ ID NO: 4; the nucleotide sequence of the downstream primer containing the HindⅢ restriction site is shown in SEQ ID NO: 5.

[0016] Further, in step S3, the vector plasmid is pET21b plasmid.

[0017] Furthermore, the competent E. coli cells are competent E. coli DH5α cells.

[0018] The present invention also provides a polyclonal antibody to glutathione S-transferase of Echinococcus multilocularis prepared by the above method, wherein the antigen of the polyclonal antibody is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1.

[0019] The invention also provides the use of the polyclonal antibody of Echinococcus multilocularis glutathione S-transferase in specifically identifying the natural glutathione S-transferase.

[0020] The invention also provides the use of the polyclonal antibody of Echinococcus multilocularis glutathione S-transferase in the preparation of anti-inflammatory drugs.

[0021] The following are the sequences involved in the scheme of the present invention:

[0022] Amino acid sequence of EmGST (SEQ ID NO: 1):

[0023] MAPTLAYWDIRGLAEQSRLLLKYLEVEYDDKRYKIGSAPTFDRSAWLSEKFSLGLDFPNLPYYIDGDFKLTQSGAILEYIADRHGMIPDCKKRRAVLHMLQCEVVDLRMA FTRTCYSPDFEKLKPGLFETLAQKLPNFEAYLGEKEWLTGDKINYPDFSLCELLNQLMKFEPTCLEKYPRLKAYLSRFENLPALRDYMASKEFKTCPCNGASAKWRGDC;

[0024] Forward primer (SEQ ID NO: 2): ATGGCTCCCACTCTGGCTTAC;

[0025] Reverse primer (SEQ ID NO: 3): CTAACAGTCACCACGCCATTTT;

[0026] Upstream primer containing NdeI restriction site (SEQ ID NO: 4):

[0027] CATATG ATGGCTCCCACTCTGGCTTAC;

[0028] Downstream primer containing HindⅢ restriction site (SEQ ID NO: 5):

[0029] AAGCTT ttaACAGTCACCACGCCATTTTG;

[0030] Nucleotide sequence of pET21b-EmGST recombinant plasmid (SEQ ID NO: 6):

[0031] AAAGGAGGCGTACATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGCTCCCACTCTGGCTTACTGGGATATCAGGGGGCTTGCGGAACAGAGTCGACTTCTGCTGAAGTACTTGGAAGTAGAGTACGATGACAAGCGATATAAGATTGGTTCGGCTCCAACTTTCGATCGTAGTGCATGGCTGTCGGAGAAGTTCTCGTTGGGTCTCGACTTTCCCAATTTGCCCTACTACATTGACGGCGACTTCAAGTTGACTCAGTCAGGGGCTATTTTGGAATATATTGCTGATAGACACGGCATGATTCCCGATTGCAAAAAGCGACGGGCAGTGCTGCACATGCTTCAATGCGAGGTTGTGGATTTGCGCATGGCGTTTACGAGGACTTGTTATAGTCCCGATTTTGAGAAGTTGAAGCCAGGTTTATTTGAGACGCTGGCACAGAAACTGCCGAACTTTGAGGCGTATTTGGGTGAGAAGGAATGGCTCACTGGTGATAAGATCAACTATCCCGACTTTAGTCTATGCGAGCTGTTGAACCAGCTGATGAAGTTTGAGCCAACGTGTCTCGAGAAGTATCCCAGACTGAAGGCCTACTTGTCGCGTTTTGAGAACTTGCCTGCATTGAGGGACTACATGGCTTCGAAGGAGTTCAAGACTTGTCCATGCAATGGAGCAAGTGCAAAATGGCGTGGTGACTGTTAAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCTCTAAACGGGGCTTTGGGGGGGTTTTTGTTGAAAGGAGGAACATATTCCGGGTTGGGGAAGGGGAGCGCCCTGTAAGGGG。

[0032] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0033] 1. Through a series of titer and specificity tests on EmGST antibodies, it is proved that the EmGST protein polyclonal antibody prepared by the recombinant protein derived from the EmGST-pET21b prokaryotic expression vector of the present invention has the advantages of high titer and good specificity. And Western blot experiments confirmed that the polyclonal antibody can specifically recognize the natural EmGST protein, clarifying that the EmGST protein is mainly expressed in the cyst wall and protoscolecus of alveolar cysts.

[0034] 2. The present invention uses EmGST-pET21b recombinant protein as an antigen to successfully prepare mouse anti-EmGST polyclonal antibodies; it also proves that EmGST protein can significantly reduce the secretion expression level of inflammatory factors IL-1β, IL-6, and TNF-α, thereby exerting an anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The results of RNA integrity verification and glutathione S-transferase gene PCR of Echinococcus multilocularis in Example 1 (M is a DNA molecular mass standard, A is total RNA of Echinococcus multilocularis; B is glutathione S-transferase gene PCR product);

[0036] Figure 2 is the pET21b-EmGST recombinant plasmid map in Example 2;

[0037] Figure 3 The expression conditions of EmGST recombinant protein induced by IPTG in Example 3 were screened (A: IPTG induction time gradient, total protein in supernatant; B: IPTG induction time gradient, total protein in precipitation (1 in A and B is the supernatant of pET21b empty vector induced for 4 hours, 2 is the supernatant of pET21b empty vector not induced, and 3 to 9 are the supernatants of pET21b-EmGST recombinant vector induced for 0, 2, 4, 6, 8, 10, and 12 hours respectively); C: IPTG induction concentration gradient, total protein in supernatant; D: IPTG induction concentration gradient, total protein in precipitation (1 in C and D is the supernatant of pET21b empty vector induced, 2 is the supernatant of pET21b empty vector not induced, and 3 to 9 are the supernatants of pET21b-EmGST recombinant vector induced with final IPTG concentrations of 0, 0.2, 0.4, 0.5, 0.6, 0.8, and 1 respectively));

[0038] Figure 4 It is the purification result of EmGST gene recombinant protein in Example 3 of the present invention (M: protein quality standard; 1-3: before purification, flow-through solution, washing solution, 4-10: eluent);

[0039] Figure 5 The ELISA method in Example 4 of the present invention detects the antibody titer of serum after EmGST immunization;

[0040] Figure 6 is the purification effect of EmGST polyclonal antibody in Example 4 of the present invention (M: protein quality standard; 1: purified EmGST polyclonal antibody);

[0041] Figure 7 The EmGST polyclonal antibody in Example 4 of the present invention recognizes the EmGST recombinant protein and the natural protein (M in A is a protein quality standard; 1 and 2 are EmGST recombinant proteins; M in B is a protein quality standard; 1, 2 and 3 are the cyst fluid, protoscolecere and cyst wall of Echinococcus multilocularis, respectively);

[0042] Figure 8 The effect of rEmGST on macrophage viability detected by CCK-8 method in Example 5 of the present invention (data are expressed as mean + SD (n = 3), compared with the control group **P < 0.01);

[0043] Fig. 9 The expression levels of pro-inflammatory factors in macrophages were detected by qRT-PCR in Example 5 of the present invention (A is the expression level of IL-6 mRNA; B is the expression level of IL-1β mRNA; C is the expression level of TNF-α mRNA; cells were pretreated with 10 μg / ml rEmGST for 24 h, and stimulated with LPS (0.5 μg / ml) for 24 h; data are expressed as mean + SD (n = 3); compared with the control group ****P < 0.0001; compared with the LPS group *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0045] It should be noted that the raw materials used in the present invention, unless otherwise specified, are conventional commercial products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0046] A method for preparing a polyclonal antibody against Echinococcus multilocularis glutathione transferase comprises the following steps:

[0047] S1. Obtain the cystic tissue of Echinococcus multilocularis, extract RNA from the cystic tissue of Echinococcus multilocularis, and prepare cDNA.

[0048] S2. Using the cDNA in step S1 as a template, reverse transcription polymerase chain reaction is used to perform PCR amplification to obtain an EmGST gene encoding an amino acid sequence as shown in SEQ ID NO: 1.

[0049] The primers used for the PCR amplification are: the forward primer is shown in SEQ ID NO: 2, and the reverse primer is shown in SEQ ID NO: 3;

[0050] The reaction system of the PCR amplification is: 2×TaqMasterMix 25μL, 10μm forward primer 2μL, 10μm reverse primer 2μL, cDNA template <0.5μg, ddH2O, Up to 50μL;

[0051] The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 25-35 cycles, extension at 72°C for 30 s, and termination extension at 72°C for 2 min.

[0052] S3, using upstream and downstream primers containing specific restriction sites to perform double enzyme reaction on the EmGST gene and plasmid, connecting the EmGST gene to the prokaryotic expression vector plasmid, constructing a recombinant vector, and then transfecting the recombinant vector into competent Escherichia coli cells, inducing prokaryotic expression and purification, to obtain the recombinant protein;

[0053] The upstream and downstream primers containing specific restriction sites are respectively an upstream primer containing an NdeI restriction site and a downstream primer containing a HindⅢ restriction site. The nucleotide sequence of the upstream primer containing an NdeI restriction site is shown in SEQ ID NO: 4; the nucleotide sequence of the downstream primer containing a HindⅢ restriction site is shown in SEQ ID NO: 5.

[0054] The vector plasmid is pET21b plasmid.

[0055] The competent E. coli cells are competent E. coli DH5α cells.

[0056] S4. Using the amino acid sequence of Echinococcus multilocularis glutathione transferase as shown in SEQ ID NO: 1 as an antigen, immunizing mice to prepare antiserum, and purifying the antiserum to obtain polyclonal antibodies against Echinococcus multilocularis glutathione transferase.

[0057] Example 1: Acquisition of the Echinococcus multilocularis glutathione transferase EmGST gene

[0058] 1.1EmGST gene query

[0059] Log in to the NCBI database and search for the full-length amino acid sequence using the EmGST accession number CAA59739.1 (https: / / www.ncbi.nlm.nih.gov / protein / CAA59739.1 / ). The nucleotide sequence of EmGST was obtained from the WormBaseParasite data, and its accession number in the Worm Base Parasite database is EmEmuJ_000538300.

[0060] By searching the NCBI protein database, the present invention successfully obtained the full-length sequence of EmGST amino acids, which was annotated as encoding glutathione transferase, also known as glutathione transferase, containing 219 amino acids, and the amino acid sequence is shown in SEQ ID NO:1.

[0061] 1.2 Obtaining Echinococcus multilocularis cyst tissue

[0062] Female SD rats (purchased from the Animal Experiment Center of Chongqing Medical University) that had been infected with Echinococcus multilocularis for six months were killed by cervical dislocation, and blood from the eyeballs was collected into EP tubes without anticoagulants. After standing at room temperature, the upper serum was separated and used for later use.

[0063] The multilocular Echinococcus cysts parasitized in the abdominal cavity of SD rats were taken out, soaked in a sterile PBS solution containing 5% penicillin / streptomycin, the cyst wall was cut, and then the cyst wall was washed with a sterile PBS solution containing 5% penicillin / streptomycin in a 50mL centrifuge tube, and the multilocular Echinococcus cyst tissue was obtained by resuspension.

[0064] 1.3 Extraction of RNA from Echinococcus multilocularis vesicle tissue

[0065] Cut 50-100 mg of Echinococcus multilocularis vesicle tissue, grind it in liquid nitrogen, add 1000ul Trizol to blow the ground tissue, and let it stand for 3 minutes. Add 200ul chloroform, shake for 15s, and let it stand at room temperature for 5 minutes. Centrifuge at 4℃12000rpm for 15min. At this time, the solution is divided into three layers. The upper layer solution is aspirated into a new centrifuge tube. Add isopropanol in a 1:1 ratio, mix well and let it stand for 5min. Centrifuge at 4℃12000rpm for 15min, pour out the supernatant, and the RNA will sink to the bottom of the tube. Add 1ml of anhydrous ethanol, mix well, and let it stand for 5min. Centrifuge at 4℃, 7500rpm for 5min, aspirate the supernatant, and retain the RNA precipitate. Open the centrifuge tube cap and dry at room temperature for 15min. After the ethanol evaporates, the RNA precipitate is translucent. Add 20ul-30ul DEPC water to dissolve it, centrifuge it instantly, and repeat 3 times to dissolve the RNA precipitate on the tube wall. The RNA concentration was measured by Nanodrop, and the RNA was labeled and stored at ultra-low temperature to obtain the RNA of the cystic tissue of Echinococcus multilocularis.

[0066] 1.3 PCR cloning of EmGST gene

[0067] 1.3.1 RT-PCR reverse transcription synthesis of cDNA: using TakaRa reverse transcription kit (Cat. No.: RR092A)

[0068] The first step is to remove genomic DNA. The total volume of the reaction system is 16 μL, including 8X gDNA Eraser PrEmix 2 μL, Echinococcus multilocularis cyst tissue RNA sample 1 μL, and RNase Free H2O 13 μL. Take an enzyme-free PCR tube, add the above reactants into it and mix well, and put it into the PCR instrument at 42°C for 2 minutes.

[0069] The second step is reverse transcription into cDNA. The total volume of the reaction system is 20 μL, including: 16 μL of the above reactants and 4 μL of 5XRTPrEmix. The above enzyme-free PCR tube is placed in the PCR instrument detection plate, 37°C, 10 min; 85°C, 5 s. Ultra-low temperature storage of cDNA.

[0070] 1.3.2 Design of PCR primers for synthesis of EmGST gene is shown in Table 1.

[0071] Table 1 PCR primer sequences and related information

[0072]

[0073] 1.3.3 Use the cDNA obtained in step 1.3.1 as a template to clone the EmGST gene.

[0074] The PCR reaction system was as follows: 2× Taq Master Mix 25 μL, 10 μm forward primer 2 μL, 10 μm reverse primer 2 μL, cDNA template <0.5 μg, ddH2O, Up to 50 μL.

[0075] Mix the above reactants and perform amplification using the following procedure:

[0076]

[0077]

[0078] PCR products were detected by 1% agarose gel electrophoresis. Figure 1 .

[0079] 1.3.5 Purify the PCR product of EmGST (using the Bio-Tech PCR Product Purification Kit (Cat. No.: D0033) to obtain the PCR purified product of EmGST. Connect it with the PMD19-T vector. The reaction system is 10 μL, including: 1 μL PMD19-T, 5 μL Solution, 1 μL PCR purified product, 3 μL RNase-Free Water. Mix the reactants, connect them at 4°C overnight, and verify their sequence.

[0080] After precise determination, the concentration of the extracted RNA solution of Echinococcus multilocularis cyst tissue was within the ideal range, and its A260 / 280 ratio was close to 2. Its purity was identified by 1% agarose gel electrophoresis, and the results showed that the total RNA of Echinococcus multilocularis cyst tissue had good integrity ( Figure 1 A), high-quality cDNA was obtained by reverse transcription, and a DNA fragment of about 660 bp was obtained after PCR amplification ( Figure 1 B), sequencing results also confirmed that the PCR sequence was consistent with the EmGST sequence.

[0081] Example 2: Construction and identification of EmGST-pET21b recombinant plasmid

[0082] 1 Experimental methods

[0083] 1.1 Design and synthesize specific primers for EmGST gene: Based on the EmGST gene sequence, upstream and downstream primers containing specific restriction sites were customized. The upstream primer sequence is: 5′- CATATG ATG GCTCCCACTCTGGCTTAC′ (where the NdeI restriction site is underlined), the downstream primer sequence is: 5′- AAGCTT ttaACAGTCACCACGCCATTTTG-3′ (the underlined part indicates the HindⅢ restriction site) These primers were precisely synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0084] 1.2 The plasmid / EmGSTPCR purified product obtained in Example 1 was subjected to double restriction endonuclease digestion reaction using restriction endonucleases NdeI and HindIII:

[0085]

[0086] After the above reactants were mixed evenly, they were first incubated in a PCR instrument at a constant temperature of 37°C for 10 minutes, and then heated to 80°C for 5 minutes to terminate the activity of the enzyme. The target fragments after enzyme digestion were then purified and recovered, and their concentration and purity were tested. The purified EmGST gene and pET21b plasmid were mixed in a ratio of 6:1, and connected using T4 ligase to construct the EmGST-pET21b recombinant vector.

[0087] 1.5 Screening and identification of positive clones

[0088] The EmGST-pET21b recombinant vector was transformed into E. coli DH5α competent cells by the heat shock transformation method, spread on LB plates containing Amp (concentration of 100 μg per ml) for culture, and positive clones were screened and sent to Sangon Biotech Co., Ltd. for sequencing verification.

[0089] 2 Experimental results

[0090] Since the pET21b plasmid has Amp resistance, the E. coli BL21 strain that has been successfully introduced with the plasmid can be selected. However, it cannot be guaranteed that all screened strains have successfully introduced EmGST-pET21. The successful construction of the EmGST-pET21b vector can be further confirmed by bacterial liquid PCR screening. In addition, the sequencing results of the pET21b-EmGST recombinant plasmid (the nucleotide sequence of the pET21b-EmGST recombinant plasmid is shown in SEQ ID NO: 6) show that the CDS region of EmGST has been successfully inserted into the expression function region of the pET21b plasmid. The map of the pET21b-EmGST recombinant plasmid is shown in Figure 2 In order to be closer to the natural state, no fusion tags were added during the construction of the EmGST gene to ensure that the subsequently expressed protein is natural and pure.

[0091] Example 3: Expression and purification of EmGST recombinant protein

[0092] 1 Experimental methods

[0093] 1.1 Analysis of the solubility of EmGST recombinant protein and screening of optimal conditions

[0094] The E. coli BL 21 expression strain with correct sequencing was selected to analyze whether it could soluble express EmGST protein, and to optimize the induction concentration and induction time of IPTG in prokaryotic expression.

[0095] 1.1.1 Screening of optimal IPTG concentration and time

[0096] (1) The correctly sequenced E. coli BL21 was inoculated on LB solid medium containing Amp (concentration of 100 μg / ml) and cultured for 14-16 h. A single colony with good growth was selected and transferred to LB liquid medium containing Amp. The culture was shaken at 37°C and 220 rpm / min until the OD600 of the bacterial solution reached 0.6-0.8.

[0097] (2) Divide the bacterial solution in step (1) into 14 tubes, each with 2 mL. In order to screen the optimal IPTG induction concentration, 7 groups of experiments were set up, using IPTG final concentrations of 0mM, 0.2mM, 0.4mM, 0.5mM, 0.6mM, 0.8mM, and 1mM, respectively, and cultured at the same temperature and speed for 5 hours. In addition, in order to explore the effect of induction time, another 7 groups of experiments were set up, with IPTG concentrations of 1mmol / L, and cultured for 0, 2, 4, 6, 8, 10, and 12 hours according to the time gradient, and the bacterial solution was collected and placed at 4°C.

[0098] 1.1.2 Extraction of EmGST recombinant protein

[0099] Centrifuge the bacterial sample in step (2) at 12,000 rpm for 10 minutes and discard the supernatant. Add 2 ml of freshly prepared lysis buffer to each tube, mix and let stand on ice for 30 minutes. After ultrasonic disruption of the bacteria, centrifuge again at 12,000 rpm at 4°C for 10 minutes. Collect the supernatant and precipitate separately, and resuspend the precipitate with an equal amount of lysis buffer to obtain the bacterial lysis buffer. Use SDS-PAGE electrophoresis to detect the target protein.

[0100] 1.2 Purification of EmGST recombinant protein

[0101] Add the bacterial lysis buffer obtained under the optimal induction conditions of two times the volume of GST purification resin, and use the elution buffer to elute the purification column. The collected eluate is the purified EmGST protein sample. After subsequent testing, several tubes of eluate with better results are mixed into one tube and stored in a refrigerator at 4°C overnight.

[0102] 2 Experimental results

[0103] 2.1 Analysis of EmGST solubility and optimization of IPTG induction conditions

[0104] Under the same IPTG concentration, the time gradient results showed that the expression of EmGST recombinant protein reached its peak after IPTG induction for 6 hours. The IPTG concentration gradient experiment found that under the same induction time, the supernatant expression of EmGST recombinant protein was most significant when IPTG reached 0.5mM. Therefore, the subsequent experiments chose to use IPTG with a final concentration of 0.5mM and an induction time of 6h. SDS-PAGE detected the EmGST recombinant protein band at around 25000 ( Figure 3 ), which is consistent with expectations.

[0105] 2.2 Purification of EmGST recombinant protein

[0106] The protein obtained under the optimal induction conditions had more impurities before purification. After passing through an affinity chromatography column containing reduced glutathione (GSH), EmGST specifically bound to GSH, while other non-specifically bound proteins were eluted. Figure 4 As shown, there is only a small amount of EmGST and most of the impurities in the flow-through. By changing the pH and ionic strength of the buffer, the EmGST fusion protein dissociates from GSH, and high-purity EmGST is eluted. The concentration of the EmGST recombinant protein is 2 mg / L as determined by the BCA protein quantification kit.

[0107] Example 4: Preparation of polyclonal antibodies against EmGST recombinant protein

[0108] 1 Experimental methods

[0109] 1.1 Immunization of BALB / C mice with EmGST recombinant protein

[0110] The purified recombinant protein of Echinococcus multilocularis glutathione transferase EmGST from Example 3 was used as an antigen and immunized BALB / C mice of about 6 to 8 weeks old by subcutaneous injection.

[0111] 1 mg / mL each time, once every 2 weeks, for a total of 2 immunizations; blood was collected for testing, and the titer of the antibody against the antigen was determined by the ELISA method. When the titer was greater than 1:500000, blood was collected from the venous plexus, and the antiserum was collected. The antiserum was purified by conventional methods to prepare polyclonal antibodies, and SDS-PAGE was performed to confirm the purification effect.

[0112] 1.2ELISA detection of serum antibody titer of immunized mice

[0113] The EmGST recombinant protein was coated in an ELISA plate (37°C, 2h) at 2ug / ml and 100ul / well. After washing with PBST, the plate was blocked overnight at 4°C with 100ul / well of blocking solution. After washing with PBST for 3 times again, the antiserum was diluted at a ratio of 1:500, and then several times of gradient dilution was performed on this basis. The plate was incubated at 37°C for 1 hour. After washing with PBST for 3 times, HRP goat anti-mouse IgG (H+L) (1:5000) was added at 100ul / well and incubated at 37°C for 45 minutes. After washing with PBST for 3 times, the substrate solution (TMB) 1 was added at 100ul / well and reacted for 5-10 minutes. Finally, 50ul / well of 2mol / L sulfuric acid was added to terminate the reaction, and the OD value of each well was measured and recorded at a wavelength of 450nm.

[0114] 1.3 Polyclonal antibody against recombinant EmGST protein recognizes native EmGST protein

[0115] Western Blot detection: The collected cyst fluid, cyst wall and natural proteins of the protoscolecere were used as key antigenic substances, and the purified BALB / C mouse serum antibodies were diluted 1:1000 to prepare the primary antibody. At the same time, the secondary antibody was diluted 1:10000. The negative control group used mouse negative serum and the secondary antibody was also diluted. The Western Blot technology was used to detect whether the serum antibody could accurately recognize the natural GST protein.

[0116] 2 Experimental results

[0117] 2.1ELISA detection of serum antibody titers of BALB / C mice after immunization

[0118] ELISA was used to detect the serum antibody titer of mice immunized with EmGST recombinant protein, and the serum of mice not immunized with EmGST recombinant protein was used as a negative control. The results of the experimental group showed a significant increase in serum antibody titer, with the highest reaching 1:512000 (e.g. Figure 5 In the negative control group (6-10 mice), the serum did not show detectable absorbance. These results fully demonstrate that the preparation of EmGST serum polyclonal antibody is ideal.

[0119] 2.2SDS-PAGE detection of serum antibody purification effect

[0120] SDS-PAGE observed that the purified EmGST mouse serum polyclonal antibody had obvious bands at 55kDa of the heavy chain and 25kDa of the light chain of mouse IgG, indicating that the antibody purification effect was good. After analysis, the accuracy of the result reached 12.5%, and Figure 6The above findings provide an important basis for further understanding and studying the characteristics of EmGST mouse serum polyclonal antibodies.

[0121] 2.3 Recognition of native EmGST by EmGST polyclonal antibody

[0122] Western Blot technology was used to detect whether EmGST polyclonal antibody could recognize EmGST recombinant protein and natural EmGST respectively. Figure 7 The results showed that a distinct specific band appeared at a molecular weight of about 25 kDa. The molecular weight of this band was consistent with the expected relative molecular weight of the EmGST recombinant protein. This showed that the EmGST polyclonal antibody could specifically recognize the EmGST recombinant protein, as well as the native EmGST protein in the cyst fluid, protoscolecere and cyst wall of Echinococcus multilocularis. This result confirmed the immunoreactivity of the EmGST recombinant protein and highlighted the specificity of the polyclonal antibody.

[0123] Example 5: Regulation of macrophage function by EmGST recombinant protein

[0124] 1 Experimental methods

[0125] 1.1 CCK-8 assay to detect the effect of rEmGST on cell viability

[0126] RAW264.7 cells were cultured, the old culture medium was removed, and 100 μL / well of cell suspension (about 5×10 3 cells), shake evenly, place in a cell culture incubator and culture for 24 hours, then discard the old culture medium, wash twice with PBS, and set up 5 groups: normal control group (cells treated with only cell culture medium), EmGST treatment group (10, 20, 40, 80, 100 μg / ml). The treatment time is 24 hours. Add 10 μL / well of CCK-8 reagent and incubate at 37°C for 2 hours; use an enzyme reader to accurately measure the absorbance at a wavelength of 450nm (see Figure 8 ).

[0127] 1.2 qRT-PCR conventional method to detect the mRNA level of pro-inflammatory factors

[0128] In order to evaluate the anti-inflammatory ability of rEmGST, RAW264.7 macrophages were treated with the control group, LPS group, and LPS+rEmGST group, and the expression levels of inflammatory factors were detected. The results are shown in Fig. 9 .

[0129] 2 Experimental results

[0130] 2.1CCK-8 test results

[0131] Figure 8 The experimental results showed that compared with the control group, the activity of macrophages was significantly improved after treatment with rEmGST at concentrations of 10, 20, 40, 80 and 100 μg / mL (P<0.01). This shows that within the test concentration range, rEmGST not only has no toxic effect on RAW264.7 cells, but may have a certain effect on promoting cell viability. This shows that rEmGST has a promoting effect on macrophage viability.

[0132] 2.2 rEmGST inhibits the production of pro-inflammatory factors

[0133] The results are as follows Fig. 9 As shown: Compared with the control group, the expression of proinflammatory factors IL-1β, TNF-α, and IL-6 was significantly increased when macrophages were stimulated with LPS alone (IL-1β((P<0.01), TNF-α(P<0.01), IL-6(P<0.01). Compared with the LPS inflammation model group, it can be found that after treatment with 10μg / ml rEmGST, the expression levels of proinflammatory factors IL-1β and IL-6 mRNA showed a significant downward trend, while the downward trend of TNF-α mRNA expression level compared with IL-10 and IL-6 was not very significant. These results indicate that 10μg / ml rEmGST can effectively inhibit the secretion of proinflammatory cytokines by RAW264.7 macrophages stimulated by LPS.

[0134] The above-described embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A glutathione S-transferase from Echinococcus multilocularis, characterized in that The amino acid sequence of the Echinococcus multilocularis glutathione S-transferase is shown in SEQ ID NO:

1.

2. A method for preparing the polyclonal antibody against glutathione S-transferase of Echinococcus multilocularis according to claim 1, characterized in that: The steps include: S1. obtaining the cystic tissue of Echinococcus multilocularis, extracting RNA from the cystic tissue of Echinococcus multilocularis, and preparing cDNA; S2, using the cDNA in step S1 as a template, performing PCR amplification by reverse transcription polymerase chain reaction to obtain an EmGST gene encoding an amino acid sequence as shown in SEQ ID NO: 1; S3, using upstream and downstream primers containing specific restriction sites to perform double enzyme reaction on the EmGST gene and plasmid, respectively, connecting the EmGST gene to the prokaryotic expression vector plasmid, constructing a recombinant vector, and then transfecting the recombinant vector into competent Escherichia coli cells, inducing prokaryotic expression and purification, and obtaining the recombinant protein; S4. Using the amino acid sequence of Echinococcus multilocularis glutathione transferase as shown in SEQ ID NO: 1 as an antigen, immunizing mice to prepare antiserum, and purifying the antiserum to obtain polyclonal antibodies against Echinococcus multilocularis glutathione transferase.

3. The method for preparing a polyclonal antibody against glutathione S-transferase of Echinococcus multilocularis according to claim 2, characterized in that: In step S2, the primers used for PCR amplification are: the forward primer is shown in SEQ ID NO: 2, and the reverse primer is shown in SEQ ID NO:

3.

4. The method for preparing a polyclonal antibody against glutathione S-transferase of Echinococcus multilocularis according to claim 2, characterized in that: In step S2, the reaction system of PCR amplification is: 2×Taq MasterMix 25μL, 10μmForward Primer 2μL, 10μmReverse Primer2μL, TEmplate cDNA<0.5μg, ddH2O, Up to 50μL; The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 25-35 cycles, extension at 72°C for 30 s, and termination extension at 72°C for 2 min.

5. The method for preparing a polyclonal antibody against glutathione S-transferase of Echinococcus multilocularis according to claim 2, characterized in that: The upstream and downstream primers containing specific restriction sites are respectively an upstream primer containing an NdeI restriction site and a downstream primer containing a HindⅢ restriction site. The nucleotide sequence of the upstream primer containing an NdeI restriction site is shown in SEQ ID NO: 4; the nucleotide sequence of the downstream primer containing a HindⅢ restriction site is shown in SEQ ID NO:

5.

6. The method for preparing a polyclonal antibody against glutathione S-transferase of Echinococcus multilocularis according to claim 2, characterized in that: In step S3, the vector plasmid is pET21b plasmid.

7. The method for preparing a polyclonal antibody against glutathione S-transferase of Echinococcus multilocularis according to claim 2, characterized in that: The competent E. coli cells are competent E. coli DH5α cells.

8. The polyclonal antibody of glutathione S-transferase of Echinococcus multilocularis prepared by the method according to any one of claims 2 to 7, characterized in that: The antigen of the polyclonal antibody is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

1.

9. Use of the polyclonal antibody against glutathione S-transferase of Echinococcus multilocularis according to claim 8 in specific recognition of natural glutathione S-transferase.

10. Use of the polyclonal antibody against glutathione S-transferase of Echinococcus multilocularis according to claim 8 in the preparation of anti-inflammatory drugs.

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