CN110564734A

CN110564734A - Recombinant fusion protein ShSAP of Egyptian schistosome and application thereof in schistosomiasis immune diagnosis

Info

Publication number: CN110564734A
Application number: CN201910929091.XA
Authority: CN
Inventors: 刘帅; 陈启军; 侯楠; 朴贤玉
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2019-09-28
Filing date: 2019-09-28
Publication date: 2019-12-13
Also published as: WO2021057003A1

Abstract

The invention discloses an Egyptian schistosome recombinant fusion protein ShSAP and application thereof in schistosomiasis immune diagnosis. The recombinant fusion protein ShSAP of Egyptian schistosome can be produced in large-scale standardized batches by a genetic engineering technology, and has stable antigen quality and good result repeatability. The recombinant fusion protein ShSAP of Egyptian schistosomiasis can be used for immune diagnosis of Egyptian schistosomiasis and immune diagnosis of Schistosoma mansoni, and has extremely high sensitivity. The recombinant fusion protein ShSAP of Egyptian schistosome of the invention has single antigen component, ShSAP-ELISA has excellent specificity to schistosomiasis immunodiagnosis, and does not generate cross reaction with serum of other parasitic disease patients, thus having wide application prospect.

Description

Recombinant fusion protein ShSAP of Egyptian schistosome and application thereof in schistosomiasis immune diagnosis

Technical Field

The invention relates to the field of biotechnology, in particular to an Egyptian schistosome recombinant fusion protein ShSAP and application thereof in schistosomiasis immune diagnosis.

Background

Schistosomiasis is an infectious parasitic disease seriously threatening human health, mainly prevails in 76 countries and regions of subfira, and the number of schistosomiasis infected people is over 2 hundred million people all over the world. The schistosomiasis parasitized on human body mainly comprises 6 types of schistosomiasis japonica, Schistosoma mansoni, Schistosoma japonicum, Schistosoma intermedia, Mega schistosoma and Schistosoma malayi, wherein the schistosomiasis japonica, Schistosoma mansoni and Schistosoma japonicum caused by the schistosoma japonicum is most widespread and most harmful. Schistosomiasis japonica is mainly prevalent in the Yangtze river basin, southeast Asia and other countries in China, schistosoma mansoni is mainly prevalent in Africa, south America and other countries, and schistosoma japonicum is mainly prevalent in Africa and the middle east. With the continuous promotion of economic globalization, medium and non-trade and foreign-aid items are increased, the number of workers and travelers is increased day by day, and the input schistosoma mansoni cases and schistosoma japonicum cases in China are on the increasing trend year by year.

At present, the diagnosis of the input schistosomiasis cases in China mostly depends on the traditional morphological detection method. The method mainly determines the disease by checking schistosome eggs in intestinal tissues, excrement or urine of a patient, not only wastes time and labor, but also has low sensitivity for diagnosing mild schistosome infected cases, and is very easy to miss-test patients with mild schistosome infection.

Compared with morphological diagnosis methods, immunological diagnosis methods have the advantages of simple and fast operation, high sensitivity and the like. However, the most commonly used antigens currently used for immunological diagnosis of schistosomiasis are schistosomiasis mixed antigen components, such as egg soluble antigen, adult soluble antigen and the like, because the crude extract antigen has complex components and is easy to cross-react with other parasite patient serum, the specificity of the schistosomiasis diagnosis method is insufficient, and the produced reagent is not suitable for standardization.

The schistosomiasis recombinant antigen produced by the genetic engineering technology has the advantages of single component, less cross reaction, large-scale batch production and the like, and is considered as an ideal schistosomiasis diagnosis antigen molecule. In recent years, Chinese scientists report a plurality of schistosomiasis japonica immunodiagnosis antigens, such as SjSP-13, SjSAPLP4 and SjSAPLP5 antigens, and Enzyme-linked immunosorbent assay (ELISA) diagnosis methods established by the antigens have extremely high sensitivity and specificity on schistosomiasis japonica diagnosis. However, because the sequence identity of the schistosoma japonicum protein and the schistosoma japonicum homologous protein has a great difference, the existing schistosoma japonicum immunodiagnosis method can not completely meet the requirement of schistosoma japonicum immunodiagnosis.

The decoding of the schistosoma japonicum genome provides unprecedented rich information resources and platforms for the research of schistosomiasis control. The invention utilizes a bioinformatics method to predict and analyze the coding gene of the whole genome of the Schistosoma japonicum, finds that a sphingolipid activator protein-like protein (ShSAP) gene family exists in the genome of the Schistosoma japonicum, and the coding protein contains one or more SapB conserved functional structural domains. The invention utilizes a gene synthesis method to carry out gene fusion on two ShSAP genes and construct a recombinant fusion protein expression vector, utilizes a prokaryotic expression system to prepare recombinant protein, and an ELISA test confirms that the obtained recombinant fusion protein has high sensitivity and specificity when being used for immunodiagnosis of the Egyptian schistosomiasis and the Schistosoma mansoni, and is a potential candidate target of an input schistosomiasis immunodiagnosis antigen, thereby completing the invention.

Disclosure of Invention

The invention aims to provide the Egyptian schistosomiasis recombinant fusion protein ShSAP and the preparation method thereof, and the invention also aims to provide the application of the Egyptian schistosomiasis recombinant fusion protein ShSAP in schistosomiasis immune diagnosis, thereby providing an input schistosomiasis immune diagnosis kit with high sensitivity and specificity.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

The invention firstly provides an Egyptian schistosome recombinant fusion protein ShSAP encoding gene optimized by an Escherichia coli codon, wherein the nucleotide sequence of the Egyptian schistosome recombinant fusion protein ShSAP encoding gene is shown as SEQ ID NO.4, and the amino acid sequence of the encoded Egyptian schistosome recombinant fusion protein ShSAP is shown as SEQ ID NO. 1.

The invention also provides a recombinant plasmid pET28a-ShSAP, which comprises the Egyptian schistosome recombinant fusion ShSAP encoding gene.

The invention also provides a coliform host cell which contains the recombinant plasmid pET28 a-ShSAP.

The invention also provides an Egyptian schistosome recombinant fusion protein ShSAP, the amino acid sequence of which is shown in SEQ ID NO. 1.

Specifically, the schistosoma japonicum recombinant fusion protein ShSAP consists of a schistosoma japonicum sphingolipid activator protein-like protein ShSAP1 and a schistosoma japonicum sphingolipid activator protein ShSAP2, wherein the signal peptide sequence of the ShSAP1 protein and the ShSAP2 protein are removed, and the amino acid sequences of the ShSAP1 protein and the ShSAP2 protein are shown in SEQ ID No.2 and SEQ ID No. 3.

the invention also provides application of the recombinant fusion protein ShSAP of Egyptian schistosomiasis in immunodiagnosis of input schistosomiasis.

In particular to a schistosomiasis ShSAP-ELISA immunodiagnosis kit, the coating antigen of which is the Egyptian schistosoma recombinant fusion protein ShSAP.

Compared with the prior art, the invention has the technical effects that:

The recombinant fusion protein ShSAP of Egyptian schistosome can be produced in large-scale standardized batches by a genetic engineering technology, and has stable antigen quality and good result repeatability. The recombinant fusion protein ShSAP of Egyptian schistosomiasis can be used for immune diagnosis of Egyptian schistosomiasis and immune diagnosis of Schistosoma mansoni, and has extremely high sensitivity. The recombinant fusion protein ShSAP of Egyptian schistosome of the invention has single antigen component, and ShSAP-ELISA has excellent specificity on schistosomiasis immunodiagnosis and does not have cross reaction with serum of other parasitic disease patients.

Drawings

In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.

FIG. 1 is a schematic structural diagram of recombinant plasmid of Schistosoma japonicum pET28a-ShSAP constructed in example 1 of the present invention.

FIG. 2 is a schematic diagram showing the result of SDS-PAGE analysis of the ShSAP recombinant protein in example 2 of the present invention. Lane M is the protein molecular weight standard, lane 1 is the control of non-induced whole bacteria, lane 2 is the whole bacteria 4 hours after induction, lane 3 is the supernatant after the lysis of the induced bacteria, lane 4 is the precipitate after the lysis of the induced bacteria, and lane 5 is the purified ShSAP recombinant protein.

FIG. 3 is a schematic diagram showing Western blotting analysis results of ShSAP recombinant protein antigen, mouse anti-His tag antibody, human sera of different schistosomiasis patients and human sera of healthy humans in example 3 of the present invention. Lane M is the protein molecular weight standard, lane 1 is the mouse anti-His tag antibody positive control, lane 2 is the serum of patients with schistosoma japonicum, lane 3 is the serum of patients with schistosoma japonicum, lane 4 is the serum of patients with schistosoma japonicum, and lane 5 is the serum of healthy human.

FIG. 4 is a diagram showing the results of the comparison of ShSAP-ELISA and SjSAP-ELISA reported in the prior art for schistosomiasis immunodiagnosis evaluation in example 5 of the present invention. A is the detection result of ShSAP-ELISA, and B is the detection result of SjSAP-ELISA.

Detailed Description

The general technical scheme of the invention is as follows:

Firstly, predicting the ShSAP gene coded by the whole genome of the schistosoma japonicum by using a bioinformatics method, then preparing the ShSAP fusion gene of the schistosoma japonicum by using a gene synthesis technology, cloning the ShSAP fusion gene to an expression plasmid vector pET-28a (+) to construct a recombinant plasmid pET28a-ShSAP of prokaryotic expression of the ShSAP fusion gene, and transforming the ShSAP fusion gene into a coliform host cell to perform ShSAP recombinant fusion protein induced expression after transformation, screening and plasmid sequencing and identification; the inclusion body protein is purified by denaturation and nickel column affinity chromatography, and finally ShSAP recombinant fusion protein is prepared. We use Westernblotting technology to verify that the prepared ShSAP recombinant fusion protein can be immunologically recognized by serum antibodies of patients with Egyptian schistosomiasis and Schistosoma mansoni. Furthermore, the preparation of schistosomiasis diagnosis reagent and kit is carried out by taking the purified ShSAP recombinant fusion protein as an immunodiagnosis antigen, and the application value of the ShSAP recombinant fusion protein in the immunodiagnosis of input schistosomiasis Egyptica and schistosoma mansoni is evaluated by ELISA technical analysis.

in order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

The present invention will be described in further detail with reference to specific examples.

Example 1 Synthesis of Egyptian Schistosoma ShSAP fusion protein Gene and construction of prokaryotic expression vector

the method utilizes a bioinformatics method to predict the ShSAP gene coded by the whole genome of the schistosoma japonicum, the amino acid sequence of the ShSAP fusion protein is shown in a sequence table SEQ ID NO.1, the ShSAP fusion protein consists of an Egyptian schistosoma japonicum sphingolipid activator protein-like protein 1 (ShSAP1) and an Egyptian schistosomiasis sphingolipid activator protein-like protein 2(ShSAP2) with signal peptide sequences removed, and the amino acid sequences of the ShSAP1 protein and the ShSAP2 protein are shown in a sequence table SEQ ID NO.2 and SEQ ID NO. 3. An ShSAP fusion protein coding gene is designed according to the codon preference of escherichia coli, then an Egyptian schistosoma ShSAP fusion gene is prepared by using a gene synthesis technology, the nucleotide sequence of the ShSAP fusion protein is shown as SEQ ID NO.4 in a sequence table, Nde I enzyme cutting sites and Xho I enzyme cutting sites are respectively designed at the 5 'end and the 3' end of the gene, and the ShSAP fusion protein coding gene is synthesized by a Suzhou Jinzhi biotechnology limited company through a manual synthesis method.

Carrying out double enzyme digestion on the artificially synthesized target gene and an expression vector pET-28a (+) by Nde I and Xho I endonucleases respectively, and recovering and purifying the gel; connecting the target gene fragment and the expression vector fragment by using T4DNA ligase, transforming the connection product into Escherichia coli Trans1-T1 competent cells, coating the transformed competent cells on an LB medium plate (containing 50 mu g/ml kanamycin), and culturing at 37 ℃ for overnight; the positive clones were picked and sent to Jinzhi Biotech, Inc., Suzhou for DNA sequencing. Sequencing analysis results show that the inserted target gene fragment has correct sequence, the recombinant plasmid pET28a-ShSAP is successfully constructed, and the recombinant plasmid map is shown in figure 1.

Example 2 prokaryotic expression and purification of Schistosoma japonicum ShSAP fusion protein

The pET28a-ShSAP recombinant plasmid with correct sequencing is transformed into expression competent cells Transetta (DE3), and the transformed competent cells are smeared on an LB medium plate (containing 50 mu g/ml kanamycin) and cultured at 37 ℃ for overnight; the positive clones were inoculated into 15mL of LB liquid medium (containing 50. mu.g/mL kanamycin), cultured overnight at 37 ℃, and the next day, 10mL of the medium was inoculated into 1L of LB medium (containing 50. mu.g/mL kanamycin), and the culture was continued until OD_600nmThe value was 0.8, and IPTG was added thereto at a final concentration of 1mM to induce expression for 4 hours, and the cells were collected by centrifugation and frozen at-80 ℃ for further use.

Re-suspending a small amount of thallus before and after induction in PBS buffer solution, adding SDS-PAGE sample buffer solution, mixing uniformly, and boiling in boiling water bath for 5min to denature protein; adding 10 μ l of the sample before and after induction into each sample loading hole respectively, and performing 12% SDS-PAGE gel electrophoresis analysis; as shown in figure 2, pET28a-ShSAP recombinant plasmid is transformed into expression competent cells, and after IPTG induction expression, an obvious expression band appears at the position of 42kDa molecular weight, and the molecular weight size of the band is consistent with the theoretical relative molecular weight of the target recombinant protein.

Resuspending the induced thallus in 40mL of bacterial lysate, ultrasonically crushing, centrifuging at 4 ℃ and 12,000rpm for 30min, and respectively collecting an inclusion body precipitate and a supernatant; and performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis on the inclusion body precipitate and the supernatant to identify the solubility of the recombinant protein. As a result, as shown in FIG. 2, the ShSAP recombinant protein was mainly present in the inclusion body pellet.

Resuspending the inclusion bodies in PBS solution containing 1% Triton-X100, ultrasonically washing for 5min, centrifuging at 12,000rpm for 15min, and collecting the inclusion body precipitate; resuspending the inclusion bodies in 8M urea, rotating and mixing the inclusion bodies evenly at 4 ℃ overnight, fully dissolving the inclusion bodies, centrifuging the mixture at 12,000rpm for 30min, and collecting supernatant; passing the supernatant through a nickel ion chelating gel column (QIAGEN) to bind the recombinant ShSAP fusion protein with 6 histidine tags to the gel column, washing the hybrid protein with 50mM imidazole, eluting the recombinant protein with 250mM imidazole, and collecting the eluate; and (4) analyzing the purified recombinant protein by SDS-PAGE gel electrophoresis to detect the protein purity. The electrophoresis result is shown in fig. 2, and the result shows that the ShSAP recombinant fusion protein with higher purity is obtained after the ShSAP column purification. The concentration of recombinant protein was determined using BCA protein quantification kit (Thermo Fisher Scientific Co.) and was performed according to the instructions.

Example 3 antigenicity detection of ShSAP recombinant protein of Egyptian schistosome

Taking 200ng ShSAP recombinant protein to load, and carrying out SDS-PAGE gel electrophoresis; transferring the protein in the PAGE gel to a PVDF membrane by adopting a wet transfer method; sealing the PVDF membrane with 5% skimmed milk powder at room temperature for 2h, and washing with TBST buffer solution for 3 times; taking mouse anti-His tag antibody as positive control and healthy human serum as negative control, respectively adding serum of patients with Egyptian schistosomiasis, serum of patients with Mandarin schistosomiasis and serum of patients with Japanese schistosomiasis (diluted by confining liquid 1: 100), incubating overnight at 4 deg.C, and washing with TBST buffer solution for 3 times; respectively adding a fluorescence-labeled anti-mouse IgG antibody or anti-human IgG antibody (diluted by a confining liquid 1:10,000), incubating for 1h at 37 ℃ in the dark, and washing for 3 times by using a TBST buffer solution; scanning and imaging by using an Odyssey infrared laser imaging system. As shown in FIG. 3, a significant band at 42kDa was recognized immunologically by mouse anti-His tag antibody, serum from patients with schistosomiasis japonica and serum from patients with schistosoma mansoni, and no band at 42kDa was recognized immunologically by serum from patients with schistosomiasis japonica and healthy human serum.

Example 4 preparation of ShSAP-ELISA immunodiagnosis kit for schistosomiasis

1) the main components of the kit comprise:

Antigen-coated solid phase carrier: ShSAP recombinant protein was diluted to 1. mu.g/mL with carbonate coating buffer (pH 9.6) and coated in polystyrene reaction wells at 100. mu.L/well.

Enzyme-labeled antibody: alkaline phosphatase-labeled goat anti-human immunoglobulin (gamma-chain specific) antibody (Sigma Co.)

Substrate: pNPP (sigma company)

Washing buffer solution: TPBS

Diluting liquid: 5g of skimmed milk powder was dissolved in 100mL of TPBS buffer.

Substrate buffer: diethanolamine 0.1mol/L (9.7ml), MgCl₂1mmol/L (10mg), adjusting pH to 9.8 with concentrated hydrochloric acid, and diluting to 100mL with distilled water.

Reaction termination solution: 12g NaOH is dissolved in distilled water, and finally the volume is fixed to 100 mL.

2) The operation procedure and detection method of the kit are as follows:

And (3) sealing: spin-drying the liquid in the wells, adding 200. mu.L/well of diluent, sealing at 37 ℃ for 1h, washing three times with PBST, 200. mu.L/well, and finally patting dry.

Adding a sample to be detected: diluting the sample serum to be detected and the diluent according to the proportion of 1:100, setting positive, negative and blank controls (only adding the sample diluent) at the same time, incubating for 30min at 37 ℃ in 100 mu L/hole.

Washing: spin-dry the well, wash three times with PBST, 200 μ L/well, and beat dry the last time.

Adding an enzyme-labeled antibody: alkaline phosphatase-labeled goat anti-human IgG (gamma-chain specific) antibody was added at a dilution of 1:5000, and the mixture was incubated at 37 ℃ for 30min, washed as above, and patted dry.

Color development: adding enzyme substrate developing solution, incubating at 37 deg.C in dark for 15min, adding stop solution, and incubating at 25 μ L/well.

Data reading and processing: OD reading with microplate reader_405nmNumber of negative2.1 times of the OD mean value of the control sample was used as a cut-off value for determining the negative or positive.

Example 5 evaluation of ShSAP-ELISA on the immunodiagnosis value of schistosomiasis

1) ShSAP-ELISA sensitivity evaluation

20 parts of serum from a pathologically confirmed blood schistosoma japonicum patient, 17 parts of serum from a blood schistosoma mansoni patient and 30 parts of serum from a blood schistosoma japonicum patient were subjected to the same ELISA procedure as in example 4, which was evaluated for sensitivity and compared with SjSAP-ELISA (Liu S, equivalent. J infection Dis.2016,214(8): 1225-34.) reported in the prior art.

The ShSAP-ELISA results are shown in Table 1 and FIG. 4A, wherein 19 of 20 sera from patients with schistosoma japonicum were positive for antibody when diagnosed by ShSAP-ELISA, and the sensitivity of ShSAP-ELISA to immunodiagnosis of schistosoma japonicum was 95.00% (95% confidence interval, 73.1% -99.7%); 17 parts of blood serum of a schistosoma mansoni patient, 15 parts of the blood serum is positive by an ShSAP-ELISA immunodiagnosis antibody, and the sensitivity of the ShSAP-ELISA to the schistosoma mansoni immunodiagnosis is 88.2 percent (95 percent confidence interval, 62.3 percent to 97.9 percent); 30 parts of serum from schistosoma japonicum patients were immunodiagnostic by ShSAP-ELISA as antibody negative.

the SjSAP-ELISA results are shown in Table 1 and FIG. 4B, 30 serum samples of schistosoma japonicum patients were all positive by SjSAP-ELISA immunodiagnosis, and the sensitivity of SjSAP-ELISA to schistosoma japonicum immunodiagnosis was 100% (95% confidence interval, 75.0% -100%); both 17 parts of serum from Schistosoma mansoni patients and 20 parts of serum from Schistosoma Egypti patients were immunodiagnostic for antibody negativity by SjSAP-ELISA.

The ShSAP-ELISA of the invention has extremely high sensitivity to the immunodiagnosis of the schistosoma mansoni and the schistosomiasis Egypti, but is not suitable for the immunodiagnosis of the schistosomiasis japonica; the SjSAP-ELISA reported in the research has extremely high sensitivity to schistosomiasis japonica immunodiagnosis, but is not suitable for schistosoma mansoni and schistosomiasis eggpanensis immunodiagnosis.

TABLE 1 comparative analysis of sensitivity of ShSAP-ELISA and SjSAP-ELISA to schistosomiasis diagnosis

2) ShSAP-ELISA specificity evaluation

50 parts of a healthy human serum sample in a non-schistosomiasis epidemic area, 19 parts of clonorchis sinensis patient serum, 22 parts of hydatid patient serum and 20 parts of plasmodium falciparum patient serum are collected, the specificity of the ShSAP-ELISA for schistosomiasis diagnosis and the cross reaction condition with other parasite patient serum are evaluated, and compared with the SjSAP-ELISA, the ELISA experimental operation is the same as that in example 4.

ShSAP-ELISA results As shown in Table 2 and FIG. 4A, 50 healthy human sera were all immunodiagnostic antibody negative by SjSAP-ELISA and ShSAP-ELISA, and the specificity of both ELISAs was 100% (95% confidence interval, 91.1% -100%); all the serum of clonorchis sinensis patients, the serum of echinococcosis hepatica patients and the serum of plasmodium falciparum patients are diagnosed as negative by the two ELISA methods, and no cross reaction occurs.

TABLE 2 comparative analysis of specificity of ShSAP-ELISA and SjSAP-ELISA for schistosomiasis diagnosis

While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are illustrative in nature and should not be construed as limiting the scope of the invention.

Claims

1. The coding gene of the Escherichia coli codon optimized recombinant fusion protein ShSAP has the nucleotide sequence shown in SEQ ID No.4, and the amino acid sequence of the coded recombinant fusion protein ShSAP is shown in SEQ ID No. 1.

2. A recombinant plasmid pET28a-ShSAP comprising the Egyptian schistosoma recombinant fusion protein ShSAP encoding gene of claim 1.

3. A coliform host cell comprising the recombinant plasmid pET28a-ShSAP of claim 2.

4. An Egyptian schistosome recombinant fusion protein ShSAP, the amino acid sequence of which is shown in SEQ ID NO. 1.

5. The recombinant fusion protein ShSAP of claim 4, which consists of the ShSAP1 protein and the ShSAP2 protein, the amino acid sequences of the ShSAP1 protein and the ShSAP2 protein are shown in SEQ ID NO.2 and SEQ ID NO. 3.

6. The recombinant fusion protein ShSAP of Egyptian schistosome as claimed in claim 4 or 5, for use in immunodiagnosis of input schistosomiasis.

7. A schistosomiasis ShSAP-ELISA immunodiagnosis kit, the coating antigen of which is the Egyptian schistosoma recombinant fusion protein ShSAP of claim 4 or 5.