WO2021057003A1

WO2021057003A1 - Schistosoma haematobium recombinant fusion protein shsap and application thereof in immunodiagnosis of schistosomiasis

Info

Publication number: WO2021057003A1
Application number: PCT/CN2020/082338
Authority: WO
Inventors: 刘帅; 陈启军; 侯楠; 朴贤玉
Original assignee: 中国医学科学院病原生物学研究所
Priority date: 2019-09-28
Filing date: 2020-03-31
Publication date: 2021-04-01
Also published as: CN110564734A

Abstract

A Schistosoma haematobium recombinant fusion protein ShSAP and an application thereof in the immunodiagnosis of schistosomiasis. The Schistosoma haematobium recombinant fusion protein ShSAP can undergo large-scale standardized mass production by means of genetic engineering technology, antigen quality is stable and the repeatability of the results is good. The fusion protein can be used for the immunodiagnosis of both schistosomiasis haematosis and schistosomiasis mansoni, and has extremely high sensitivity. The Schistosoma haematobium recombinant fusion protein ShSAP has a single antigen component, and ShSAP-ELISA has excellent specificity for the immunodiagnosis of schistosomiasis. The fusion protein does not cross-react with the serum of a patient suffering from another parasitic disease, and has broad application prospects.

Description

A Schistosomiasis recombinant fusion protein ShSAP and its application in immunodiagnosis of schistosomiasis

Technical field

The invention relates to the field of biotechnology, in particular to a Schistosoma haematobium recombinant fusion protein ShSAP and its application in immunodiagnosis of schistosomiasis.

Background technique

Schistosomiasis is an infectious parasitic disease that seriously threatens human health. It is mainly prevalent in 76 countries and regions in Asia, Africa and Latin America. The number of people infected with schistosomiasis in the world exceeds 200 million. There are six main species of schistosomes that parasitize the human body, including Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma intervening, Schistosoma mekongi, and Schistosoma malay. Among them, Schistosomiasis caused by Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematob The widest is the most harmful. Schistosomiasis japonicum is mainly endemic in the Yangtze River Basin and Southeast Asia and other countries, Schistosoma mansoni is mainly endemic in Africa and South America and other countries, and Schistosoma haematobium is mainly endemic in Africa and the Middle East. With the continuous advancement of economic globalization and the implementation of my country’s “One Belt One Road” development strategy, China-Africa trade and foreign aid projects have increased, and the number of overseas workers and travelers has increased day by day. Imported cases of schistosomiasis mansoni and schistosomiasis in my country are increasing year by year. .

At present, the diagnosis of imported schistosomiasis cases in my country mostly relies on traditional morphological detection methods. This method mainly diagnoses the disease by examining the schistosome eggs in the patient's intestinal tissue, feces or urine. It not only takes time and effort, but also has low sensitivity in the diagnosis of mild schistosomiasis infection, and it is easy to miss patients with mild schistosomiasis infection.

Compared with morphological diagnosis methods, immunological diagnosis methods have the advantages of simple and quick operation and high sensitivity. However, the most commonly used antigen for immunological diagnosis of schistosomiasis is the mixed antigen component of schistosomes, such as egg soluble antigen, adult soluble antigen, etc. Because the composition of this crude antigen is complex, it is easy to combine with other antigens. The cross-reaction of the sera of parasite patients resulted in insufficient specificity of this type of schistosomiasis diagnosis method, and the reagents produced should not be standardized.

Relying on genetic engineering technology to produce schistosomiasis recombinant antigen molecule has the advantages of single component, less cross-reaction, large-scale mass production, etc., and is considered to be an ideal schistosomiasis diagnostic antigen molecule. In recent years, Chinese scientists have reported a number of schistosomiasis immunodiagnostic antigens, such as SjSP-13, SjSAPLP4 and SjSAPLP5 antigens. Enzyme-linked immunosorbent assay (ELISA) diagnostic methods established with these antigens It has extremely high sensitivity and specificity for the diagnosis of schistosomiasis japonica. However, due to the large differences in the sequence of the Schistosoma japonicum protein and the homologous protein of Schistosoma haematobium, the existing immunodiagnostic methods for schistosomiasis can not fully meet the needs of immunodiagnosis of schistosomiasis.

The deciphering of the schistosomiasis genome has provided an unprecedented rich information resource and platform for the research of schistosomiasis control. The present invention uses bioinformatics methods to predict and analyze the coding genes of the Schistosoma haematobium genome and find that there is a sphingolipid activation protein-like protein (Schistosoma haematobium Saposin-like protein, ShSAP) gene family in the Schistosoma haematobium genome, and the coding protein contains one or more genes. A conserved functional domain of SapB. The present invention uses gene synthesis method to genetically fuse two of the ShSAP genes and constructs a recombinant fusion protein expression vector, and uses a prokaryotic expression system to prepare the recombinant protein. The ELISA test confirms that the obtained recombinant fusion protein is used for schistosomiasis hig and Schistosomiasis mansoni The diagnosis has high sensitivity and specificity, and is a potential candidate target of immunodiagnostic antigen for imported schistosomiasis, thus completing the present invention.

Summary of the invention

The object of the present invention is to provide a Schistosoma haematobium recombinant fusion protein ShSAP and a preparation method thereof. Another object of the present invention is to provide an application of Schistosoma haematobium recombinant fusion protein ShSAP in the immunodiagnosis of schistosomiasis. A highly sensitive and specific immunodiagnostic kit for imported schistosomiasis.

In order to achieve the above objectives, the technical solutions provided by the present invention are as follows:

The present invention first provides a Schistosoma haematobium recombinant fusion protein ShSAP encoding gene optimized by E. coli codons. Its nucleotide sequence is shown in SEQ ID NO. 4, and the amino acid sequence of the Schistosoma haematobium recombinant fusion protein ShSAP encoded by it is shown as SEQ ID NO.1 is shown.

The present invention also provides a recombinant plasmid pET28a-ShSAP, which contains the aforementioned Schistosoma haematobium recombinant fusion ShSAP coding gene.

The present invention also provides a host cell of Escherichia coli, which comprises the above-mentioned recombinant plasmid pET28a-ShSAP.

The present invention also provides a Schistosoma haematobium recombinant fusion protein ShSAP, the amino acid sequence of which is shown in SEQ ID NO.1.

Specifically, the aforementioned Schistosoma haematobium recombinant fusion protein ShSAP is composed of Schistosoma haematobium sphingolipid activator protein-like protein ShSAP1 and Schistosoma haematobium sphingolipid activator protein-like protein ShSAP2 with the signal peptide sequence removed, and the amino acid sequences of the ShSAP1 protein and the ShSAP2 protein are as SEQ ID NO.2 and SEQ ID NO.3 are shown.

The invention also provides the application of the above-mentioned Schistosoma haematobium recombinant fusion protein ShSAP in the immunodiagnosis of imported schistosomiasis.

Specifically, a ShSAP-ELISA immunodiagnostic kit for schistosomiasis is provided, the coating antigen of which is the aforementioned Schistosoma haematobium recombinant fusion protein ShSAP.

Compared with the prior art, the technical effect of the present invention:

The Schistosoma haematobium recombinant fusion protein ShSAP of the present invention can be produced in large-scale standardized batches through genetic engineering technology, with stable antigen quality and good reproducibility. The recombinant fusion protein ShSAP of Schistosoma haematobium of the present invention can be used for both immunodiagnosis of schistosomiasis haematobium and schistosomiasis mansoni, and has extremely high sensitivity. The recombinant fusion protein ShSAP of Schistosoma haematobium of the present invention has a single antigen component, and ShSAP-ELISA has excellent specificity for immunodiagnosis of schistosomiasis, and does not cross-react with the serum of patients with other parasitic diseases.

Description of the drawings

In order to explain the embodiments of the present application or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the embodiments. Obviously, the drawings in the following description are only recorded in the present invention. For some of the embodiments of, for those of ordinary skill in the art, other drawings may be obtained based on these drawings.

Figure 1 is a schematic structural diagram of the recombinant plasmid pET28a-ShSAP of Schistosoma haematobium constructed in Example 1 of the present invention.

Figure 2 is a schematic diagram of SDS-PAGE analysis results of ShSAP recombinant protein in Example 2 of the present invention. Lane M is the protein molecular weight standard, Lane 1 is the uninduced whole bacteria control, Lane 2 is the whole bacteria 4 hours after induction, Lane 3 is the supernatant after the induction of bacterial lysis, Lane 4 is the precipitation after induction of the bacterial lysis, Lane 5 is the purified ShSAP recombinant protein.

3 is a schematic diagram of Western blotting analysis results of ShSAP recombinant protein antigen and mouse anti-His tag antibody, serum of different schistosomiasis patients and healthy human serum 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 human serum from patients with schistosomiasis, lane 3 is serum from patients with Schistosoma mansoni, lane 4 is serum from patients with Schistosoma japonicum, lane 5 is healthy Human serum.

Fig. 4 is a schematic diagram of the comparison between ShSAP-ELISA in Example 5 of the present invention and SjSAP-ELISA used in immunodiagnosis evaluation of schistosomiasis. A is the test result of ShSAP-ELISA, and B is the test result of SjSAP-ELISA.

detailed description

The overall technical scheme of the present invention is as follows:

First, we use bioinformatics methods to predict the ShSAP gene encoded by the Schistosoma haematobium genome, and then use gene synthesis technology to prepare the Schistosoma haematobium ShSAP fusion gene, and clone it into the expression plasmid vector pET-28a(+) to construct the Schistosoma haematobium ShSAP fusion gene Prokaryotic expression of recombinant plasmid pET28a-ShSAP, after transformation screening, plasmid sequencing and confirmation, transformed into E. coli host cells to induce expression of ShSAP recombinant fusion protein; inclusion body protein was purified by denaturation and nickel column affinity chromatography, and finally Prepare ShSAP recombinant fusion protein. We use Western blotting technology to verify that the prepared ShSAP recombinant fusion protein can be immunorecognized by sera antibodies from patients with schistosomiasis and schistosomiasis. Furthermore, we used the purified ShSAP recombinant fusion protein as the immunodiagnostic antigen to prepare schistosomiasis diagnostic reagents and kits, and evaluated its application value in the immunodiagnosis of imported schistosomiasis and schistosomiasis mansoni through ELISA technology analysis.

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

The present invention will be further described in detail below in conjunction with specific embodiments.

Example 1 Synthesis of ShSAP fusion protein gene of Schistosoma haematobium and construction of prokaryotic expression vector

The ShSAP gene encoded by the whole genome of Schistosoma haematobium was predicted by bioinformatics methods. The amino acid sequence of the Schistosoma haematobium ShSAP fusion protein is shown in SEQ ID NO.1 in the sequence table. The ShSAP fusion protein is activated by the sphingolipid of Schistosoma haematobium with the signal peptide sequence removed. Protein-like protein 1 (ShSAP1) and Schistosoma haematobium sphingolipid-activating protein-like protein 2 (ShSAP2) are composed. The amino acid sequences of ShSAP1 protein and ShSAP2 protein are as shown in SEQ ID NO. 2 and SEQ ID NO. 3 in the sequence table. According to the codon preference of Escherichia coli, the ShSAP fusion protein encoding gene was designed, and then the Schistosoma haematobium ShSAP fusion gene was prepared by gene synthesis technology. Its nucleotide sequence is as shown in SEQ ID NO. 4 in the sequence table, and at the same time in gene 5. The Nde I and Xho I restriction sites were designed on the'end and 3'end respectively, and the ShSAP fusion protein encoding gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. through artificial synthesis.

The artificially synthesized target gene and expression vector pET-28a(+) were digested with Nde I and Xho I endonucleases respectively, and the gel was recovered and purified; T4 DNA ligase was used to connect the target gene fragment and the expression vector fragment, and The ligation product was transformed into E. coli Trans1-T1 competent cells, and the transformed competent cells were spread on an LB medium plate (containing 50μg/ml kanamycin) and cultured overnight at 37℃; positive clones were picked and sent to Suzhou Jinweizhi Biotech Co., Ltd. conducts DNA sequencing. Sequencing analysis results showed that the inserted target gene fragment sequence was correct, the recombinant plasmid pET28a-ShSAP was successfully constructed, and the recombinant plasmid map is shown in Figure 1.

Example 2 Prokaryotic expression and purification of Schistosoma haematobium ShSAP fusion protein

Transform the correctly sequenced pET28a-ShSAP recombinant plasmid to express competent cells Transetta (DE3), apply the transformed competent cells to LB medium plates (containing 50μg/ml kanamycin), and incubate overnight at 37°C; The positive clones were inoculated into 15 mL of LB liquid medium (containing 50μg/ml kanamycin), cultured overnight at 37°C, and the next day, 10 mL of medium was transferred to 1L LB medium (containing 50μg/ml kanamycin). Continue to culture until the OD _600nm value is 0.8, then add IPTG with a final concentration of 1 mM to induce expression for 4 hours, collect the bacteria by centrifugation, and freeze at -80°C for later use.

Take a small amount of pre-induction and post-induction bacteria and suspend them in PBS buffer, add SDS-PAGE loading buffer, mix well and boil in a boiling water bath for 5 minutes to denature the protein; add pre-induction and pre-induction to each loading well. 10μl each of the induced samples were analyzed by 12% SDS-PAGE gel electrophoresis; as shown in Figure 2, the pET28a-ShSAP recombinant plasmid was transformed into competent cells for expression, and an obvious expression bar appeared at the molecular weight position of 42kDa after IPTG induced expression Band, its molecular weight is consistent with the theoretical relative molecular weight of the target recombinant protein.

Suspend the induced bacteria body in 40mL bacterial lysate, sonicate it, centrifuge at 4℃, 12,000rpm for 30min, collect the inclusion body precipitate and supernatant respectively; subject the inclusion body precipitate and supernatant to SDS-PAGE gel electrophoresis Analyze and identify the solubility of the recombinant protein. The results are shown in Figure 2. The ShSAP recombinant protein mainly exists in the precipitation of inclusion bodies.

Suspend the inclusion body in a PBS solution containing 1% Triton-X100 for 5 min ultrasonically, and centrifuge at 12,000 rpm for 15 min to collect the inclusion body precipitate; resuspend the inclusion body in 8M urea, rotate and mix overnight at 4°C, fully dissolve the inclusion body, and centrifuge at 12,000 rpm Collect the supernatant in 30 minutes; pass 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, wash the mixed protein with 50 mM imidazole, and then use The recombinant protein was eluted with 250mM imidazole, and the eluate was collected; the purified recombinant protein was analyzed by SDS-PAGE gel electrophoresis to detect the protein purity. The results of electrophoresis are shown in Figure 2, and the results show that the ShSAP recombinant fusion protein with higher purity is obtained after purification by a nickel ion chelating gel column. Use the BCA protein quantification kit (Thermo Fisher Scientific) to determine the concentration of the recombinant protein, and follow the instructions.

Example 3 Antigenicity Detection of ShSAP Recombinant Protein of Schistosoma haematobium

200ng of ShSAP recombinant protein was loaded and subjected to SDS-PAGE gel electrophoresis; the protein in the PAGE gel was transferred to the PVDF membrane by wet transfer method; the PVDF membrane was blocked with 5% skimmed milk powder at room temperature for 2 hours, and washed 3 times with TBST buffer; With mouse anti-His tag antibody as positive control and healthy human serum as negative control, sera from patients with schistosomiasis, sera from patients with schistosomiasis mansoni, and patients with schistosomiasis (diluted 1:100 with blocking solution) were added, respectively, at 4°C Incubate overnight, wash 3 times with TBST buffer; add fluorescently labeled anti-mouse IgG antibody or anti-human IgG antibody (diluted with blocking solution 1:10,000), incubate at 37°C in the dark for 1 hour, and wash 3 times with TBST buffer; use Odyssey infrared laser imaging system scanning imaging. The results are shown in Figure 3, there is a clear band at 42kDa that can be immunorecognized by mouse anti-His tag antibody, sera from patients with schistosomiasis and sera from patients with schistosomiasis. The sera from patients with schistosomiasis and healthy humans are at 42kDa. There is no immune recognition band.

Example 4 Preparation of ShSAP-ELISA immunodiagnostic kit for schistosomiasis

1) The main components of the kit include:

Antigen-coated solid-phase carrier: Dilute ShSAP recombinant protein to 1μg/mL with carbonate coating buffer (pH 9.6), and coat it in polystyrene reaction wells, 100μL/well.

Enzyme-labeled antibody: goat anti-human immunoglobulin (γ-chain specific) antibody labeled with alkaline phosphatase (Sigma)

Substrate: pNPP (sigma company)

Washing buffer: TPBS

Diluent: 5g skimmed milk powder dissolved in 100mL of TPBS buffer.

Substrate buffer: diethanolamine 0.1mol/L (9.7ml), MgCl ₂ 1mmol/L (10mg), concentrated hydrochloric acid to adjust the pH to 9.8, distilled water to make the volume to 100mL.

Reaction termination liquid: 12g of NaOH dissolved in distilled water, and finally the volume to 100mL.

2) The operating procedures and detection methods of the kit:

Blocking: spin-dry the liquid in the well, add 200μL/well of diluent, block for 1h at 37°C, wash three times with PBST, 200μL/well, and pat dry for the last time.

Add sample to be tested: Dilute the serum and diluent of the sample to be tested at a ratio of 1:100, and set up positive, negative and blank controls (only add sample diluent), 100μL/well, and incubate at 37°C for 30min.

Washing: Spin dry the liquid in the well, wash three times with PBST, 200μL/well, pat dry the last time.

Add enzyme-labeled antibody: Add 1:5000 diluted alkaline phosphatase-labeled goat anti-human IgG (γ-chain specific) antibody, 100μL/well, incubate at 37°C for 30min, wash as above, and pat dry.

Color development: add enzyme substrate color development solution, 100μL/well, incubate at 37°C in the dark for 15min, add stop solution, 25μL/well.

Data reading and processing: Use a microplate reader to read the OD _405nm value, and use 2.1 times the average OD of the negative control sample as the cut-off for judging negative or positive.

Example 5 Evaluation of the value of ShSAP-ELISA in the immunodiagnosis of schistosomiasis

1) ShSAP-ELISA sensitivity evaluation

Etiologically confirmed 20 sera of patients with Schistosoma haematobium, 17 sera from patients with Schistosoma mansoni and 30 sera from patients with Schistosoma japonicum. The sensitivity of ShSAP-ELISA was evaluated and compared with the previously reported SjSAP-ELISA [Liu S,et al. J Infect Dis. 2016,214(8):1225-34.] For comparison, the ELISA experiment operation was the same as in Example 4.

The results of ShSAP-ELISA are shown in Table 1 and Figure 4A. Of the 20 sera from patients with schistosomiasis, 19 sera were diagnosed as antibody positive by ShSAP-ELISA. The sensitivity of ShSAP-ELISA for immunodiagnosis of schistosomiasis was 95.00% (95%). Confidence interval, 73.1%-99.7%); out of 17 sera from patients with schistosomiasis mansoni, 15 sera were immunodiagnosed as antibody positive by ShSAP-ELISA. The sensitivity of ShSAP-ELISA for immunodiagnosis of schistosomiasis was 88.2% (95%). Confidence interval, 62.3%-97.9%); 30 sera from patients with Schistosoma japonicum were immunodiagnosed as antibody negative by ShSAP-ELISA.

The results of SjSAP-ELISA are shown in Table 1 and Figure 4B. 30 sera from patients with schistosomiasis japonicum were all immunodiagnosed as antibody positive by SjSAP-ELISA. The sensitivity of SjSAP-ELISA for immunodiagnosis of schistosomiasis japonica was 100% (95% confidence interval, 75.0%-100%); 17 sera from patients with Schistosoma mansoni and 20 sera from patients with Schistosoma haematobium were all diagnosed as antibody negative by SjSAP-ELISA.

The ShSAP-ELISA of the present invention has extremely high sensitivity for the immunodiagnosis of schistosomiasis mansoni and schistosomiasis, but is not suitable for the immunodiagnosis of schistosomiasis japonica; the SjSAP-ELISA that has been researched and reported is extremely high for the immunodiagnosis of schistosomiasis japonica The sensitivity is high, but it is not applicable to the immunodiagnosis of schistosomiasis mansoni and schistosomiasis.

Table 1 Comparative analysis of the sensitivity of ShSAP-ELISA and SjSAP-ELISA to schistosomiasis diagnosis

2) Evaluation of the specificity of ShSAP-ELISA

Collect 50 sera samples from healthy people in non-schistosomiasis endemic areas, 19 sera from patients with clonorchiasis, 22 sera from patients with hepatic hydatid disease, and 20 sera from patients with falciparum malaria to evaluate the specificity of ShSAP-ELISA for the diagnosis of schistosomiasis , And the cross-reactivity with the sera of patients with other parasitic diseases, and compared with SjSAP-ELISA. The ELISA experiment operation is the same as in Example 4.

The results of ShSAP-ELISA are shown in Table 2 and Figure 4A. 50 healthy human sera were all immunodiagnosed as antibody negative by SjSAP-ELISA and ShSAP-ELISA. The specificity of the two ELISAs was 100% (95% confidence interval, 91.1%). -100%); all serum from patients with clonorchiasis, sera from patients with hepatic hydatid disease, and sera from patients with falciparum malaria were diagnosed as negative by the two ELISAs, and there was no cross reaction.

Table 2 Comparative analysis of the specificity of ShSAP-ELISA and SjSAP-ELISA for the diagnosis of schistosomiasis

Certain exemplary embodiments of the present invention have been described above only by way of illustration. Needless to say, for those of ordinary skill in the art, without departing from the spirit and scope of the present invention, various different ways can be used to The described embodiment is modified. Therefore, the above-mentioned drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

An E. coli codon-optimized Schistosoma haematobium recombinant fusion protein ShSAP encoding gene, its nucleotide sequence is shown in SEQ ID NO.4, and the amino acid sequence of the Schistosoma haematobium recombinant fusion protein ShSAP encoded by it is shown in SEQ ID NO.1 Shown.
A recombinant plasmid pET28a-ShSAP, which comprises the Schistosoma haematobium recombinant fusion protein ShSAP encoding gene of claim 1.
A coliform host cell comprising the recombinant plasmid pET28a-ShSAP of claim 2.
A Schistosoma haematobium recombinant fusion protein ShSAP, whose amino acid sequence is shown in SEQ ID NO.1.
The Schistosoma haematobium recombinant fusion protein ShSAP according to claim 4, which is composed of Schistosoma haematobium sphingolipid activator protein-like protein ShSAP1 and Schistosoma haematobium sphingolipid activator protein-like protein ShSAP2 with the signal peptide sequence removed, and the amino acid sequences of the ShSAP1 protein and the ShSAP2 protein As shown in SEQ ID NO.2 and SEQ ID NO.3.
The Schistosoma haematobium recombinant fusion protein ShSAP according to claim 4 or 5, and its application in the immunodiagnosis of imported schistosomiasis.
A ShSAP-ELISA immunodiagnostic kit for schistosomiasis, the coating antigen of which is the recombinant fusion protein ShSAP of Schistosoma haematobium according to claim 4 or 5.