CN101205538B - Clone, expression and use of Schistosoma Japonicum signal transduction protein Sjwnt-4 gene - Google Patents

Abstract

The invention discloses a Wnt family gene which is amplified for the first time from Schistosoma japonicum 19-day schistosomulum by utilization of RACE technology, wherein, sequence analysis indicatesthat: a complete coding frame of the gene comprises 1311bp, 436 amino acids coded; and theoretical molecular weight of 49.6kD. Homology analysis results indicate that: amino acid sequences of the gen e have typical Wnt family protein characteristics; similarity of the amino acid sequences with amino acid sequences of Dugesia japonica and human Wnt4 is relatively 43 percent and 37 percent; the geneis presumed to be a Wnt4 gene of schistosome and named as Sjwnt4 (GenBanK Log-On No.: DQ643829). Real-time quantitative PCR analysis indicates that: the gene is expressed all in 14-day schistosomulu m, 19-day schistosomulum, 31-day imago, 44-day female worms and 44-day male worms. The invention constructs a pronucleus expression vector pGEX-4T-2-Sjwnt4 of the gene and applies an Escherichia coli system for expression; expression proteins exist in the form of inclusion bodies; Western blottings indicate that expression products can be identified by crude antigen immune serums of Schistosoma japonicum imago.

Description

Cloning, expression and application of signal transduction protein Sjwnt-4 gene of Schistosoma japonicum

Technical field:

The invention belongs to the field of gene technology, and in particular relates to the cloning, expression and application of a Japanese blood sucking signal transduction protein Sjwnt-4 gene.

Background technique:

Wnts are a kind of secreted glycoproteins that can be found in hydra, insects and vertebrates. After being secreted by cells, they bind to membrane receptors of themselves or adjacent cells, stimulate downstream signaling pathways, and regulate the expression of target genes in the nucleus. Expression, determines the differentiation fate of cells, and plays a broad regulatory role in animal development. Abnormal spatiotemporal expression or abnormal activation of Wnt protein is often related to the occurrence of tumors. Studies have shown that the Wnt signaling pathway plays a key role in regulating the normal development of the mammalian reproductive system. It is mainly involved in the formation of Mullerian ducts and their derived organs, and regulates the development of follicles, ovulation and luteinization. In addition, it is related to normal pregnancy. The establishment of breast cancer and changes in the mammary gland during pregnancy are also related. Wnt4 is one of the members of the Wnt family protein. In mice, Wnt4 plays a key role in the formation of Müllerian tubes. Both male and female mice lacking Wnt4 have no Müllerian tubes; female mice with Wnt4 mutations have no Müllerian tubes. The derived organs of the tubes (i.e., fallopian tubes, uterus, cervix, and upper vagina); whereas the Wolffian tubes continue to exist. The development of the testes of male animals lacking Wnt4 is not affected, but once the Wnt4 signal is lost in the developing female gonad, its structure and function show certain masculinization characteristics. Although the gonads are located in their correct parts, their The ovary is rounder than normal and uncoated like a testicle. In addition, mutant gonads can differentiate MIS (Müllerian inhibitory substance) and testosterone. In humans, the diploidy of the chromosome 1p31-p35 segment in males leads to the increase of Wnt4, and the overexpression of Wnt4 causes XY males to have a female phenotype. Therefore, Wnt4 may be a sex phenotype-determining gene.

The research on the growth and development signal transduction of schistosomiasis is an effective way to understand and understand the life characteristics of schistosomiasis. But so far, little research has been done on the signal transduction system of growth and development of schistosomiasis, resulting in slow progress in developing highly effective candidate vaccine molecules and screening new drugs. However, there is no research report on the Wnt gene of Schistosoma japonicum at home and abroad.

Invention content:

The technical problem to be solved by the invention is to design the relevant expression of the Wnt gene of the schistosome.

In the present invention, the ORF-containing cDNA sequence encoding Schistosoma japonicum Wnt4 protein is cloned for the first time by RACE technology, the expression of the gene in different developmental stages of Schistosoma japonicum is analyzed, and the expressed product is expressed by Escherichia coli, and the antigenicity of the expressed product is tested Determination.

The invention provides the cloning and expression of a Schistosoma japonicum signal transduction protein Sjwnt-4 gene.

The method includes the following steps:

(1) Material

Trizol and GeneRacer ^™ Kit were purchased from Invitrogen; Ex Taq Hot Start DNA polymerase, restriction endonuclease BamH I, Xho I, T4 DNA ligase, fluorescent quantitative PCR detection kit and random primers were purchased from TaKaRa Bioengineering (Dalian) Co., Ltd.; goat anti-mouse IgG-HRP was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; anti-GST monoclonal antibody was purchased from SIGMA; goat anti-rabbit IgG-HRP was purchased from Huamei Bioengineering Company; reverse transcriptase was purchased from Stratagen ; RNase inhibitors were purchased from Promega; SYBR GreenI and Calibration were purchased from BIO-RAD;

(2) Strains, plasmids and experimental animals

Plasmid pGEX-4T-2, Escherichia coli DH5α, BL21(DE3) were provided by our institute (Shanghai Veterinary Research Institute), and pUCm-T vector was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. New Zealand white rabbits (male, 2.5-3.0 kg) were purchased from Shanghai Luojing Feida Experimental Animal Farm. The cercariae of the mainland China strain of Schistosoma japonicum were provided by the snail laboratory of our institute (Shanghai Veterinary Research Institute). New Zealand white rabbits were infected with 20 000, 5000, and 2000 cercariae of Schistosoma japonicum by abdominal patch method, and were killed after 14 days, 19 days, 31 days and 44 days, and the worms were collected by hepatic portal vein perfusion and frozen in liquid nitrogen for later use;

(3) method

① Extraction of total RNA Take 200 mg of Schistosoma japonicum 19-day-old larvae frozen in liquid nitrogen, and extract total RNA according to the instructions of the Trizol kit;

② Primer design and amplification of RACE In our laboratory, Cheng Guofeng and others used two-dimensional electrophoresis combined with peptide mass fingerprinting technology to obtain a peptide sequence of a Wnt family protein, and used this peptide sequence as a query sequence in the Schistosoma japonicum EST library (http://www .ncbi.nlm.nih.gov/BLAST) found a corresponding EST fragment of Schistosoma japonicum (GenBank ^TM accession number AAM89872), which is 727bp long and does not contain a complete ORF. Using the EST sequence as a template, RACE primers (synthesized by Shanghai Shenneng Bocai Biotechnology Co., Ltd.) were designed and synthesized. Two nested primers for 3′RACE: 3GSP1: 5′-TATGCTGTGGTCGAGGATTTAAACG-3′, 3GSP2: 5′-GGTGTTGCAAAGTTGTCTGTAAAACA-3′; Two nested primers for 5′RACE: 5GSP1: 5′-GGTGTTGCAAAGTTGTCTGTAAAACA-3′, 5GSP2: 5'-TTGTCGTTTAAATCCTCGACCACAG-3'; the specific steps were performed according to the operation manual of the GeneRacerTM kit. The RACE product was cloned into the pCR4-TOPO vector provided by the kit, and positive clones were selected for sequencing by Handsome Biotechnology Co., Ltd. Use DNAStar software to find the overlap between the 3′ and 5′ RACE product sequences and the original EST sequence, and splice the three sequences;

③Amplification of cDNA fragments containing ORF According to the spliced cDNA sequence, primers (F1 and F2) were designed to amplify cDNA fragments containing ORF. F1: 5′-CCG GGATCC ATGAATCTAACTCAGCTAGAA-3′ introduced restriction site BamHI (underlined); F2: 5′-CGG CTCGAG TTAATTACATGTAGATATAAC-3′ introduced restriction site (XhoI) (provided by Shanghai Shenergy Bocai Biotechnology Ltd Synthetics). Take 3 μl of cDNA obtained by reverse transcription in 3′RACE as a template for PCR amplification. The PCR reaction conditions are pre-denaturation at 94°C for 5 minutes, followed by 94°C, 30s, 55°C, 30s, 72°C, 1min30s, a total of 30 Cycle, and extend at 72°C for 10 min after the cycle ends. The PCR product was recovered with an agarose gel DNA recovery kit. After purification, it was connected to the pUCm-T vector and transformed into E. coli DH5α competent cells. A single colony was selected for enzyme digestion and identification. The positive plasmid was named pUCm-T-Sjwnt4 and sent to Yingjun Biotechnology Limited Sequencing;

④Bioinformatics analysis Perform similarity comparison of the sequenced cDNA on NCBI (http://www.ncbi.nlm.nih.gov/BLAST); use ORF finder on NCBI to find the reading frame of the new gene ; Use DNAstar software to analyze parameters such as the number of amino acid residues, composition, and protein relative molecular weight; use clustalW software to perform multiple comparisons of Wnt proteins in different species; use NetAcet software (http://www.cbs.dtu.dk/services /NetAcet/) for glycosylation site prediction. The MHCII phenotype online prediction tool of SYFPEITHI (http://WWW.uni-tuebingen.de/uni/kxi/) was used for T cell epitope prediction.

⑤In the fluorescence real-time quantitative RT-PCR test, the housekeeping gene α-tubulin of Schistosoma japonicum was selected as the internal reference; the total RNA of 14d and 19d larvae, 31d worms, 44d male worms and 44d female worms were extracted respectively, and After genomic DNA, use random primers to synthesize the first strand of cDNA; fluorescent quantitative PCR primers: primers for amplifying Sjwnt4: S1: 5'-ACATACAAAATCGTCTGGTCTC-3', S2: 5'-GATGGTAAAGGCGATGTAGTC-3', the length of the amplified fragment is 214bp; Add α-tubulin primers: T1: 5′-CTGATTTTCCATTCGTTTG-3′, T2: 5′-GTTGTCTACCATGAAGGCA-3′, the length of the amplified fragment is 213bp; the primers were synthesized by Shanghai Sangong, and the fluorescent dye method was used for real-time quantitative PCR. The reaction system Contains: 5×R-PCR Buffer 5μl, 250mM Mg ²⁺ 0.3μl, 10mM dNTP 0.75μl, 10μM primer 1.0μl, 25×SYBR Green I 1.0μl, 10-3×Calibration 1.0μl, 5U/μl Ex Taq Hs 0.25μl , ddh ₂ O 14.7 μl, template cDNA 1.0 μl, a total of 25 μl; reaction parameters: denaturation at 95°C for 3 minutes, 95°C for 5s, 58°C for 30s, 40 cycles, the end time point at 58°C for 30s is the detection point of fluorescence signal, each All reactions were repeated in 3 wells; BIO-RAD's iCycler ^TM IQ version 3.0 software was used for calculation and analysis, and α-tubulin was used as the internal reference to standardize the reaction results to obtain the content of the target gene relative to every million housekeeping genes;

⑥Construction of recombinant expression plasmid From the recombinant plasmid pUCm-T-Sjwnt4 determined after sequencing, use restriction endonucleases BamHI and Xho I to cut out the cDNA fragment of Sjwnt4 containing ORF, and clone the complete sequence into the prokaryotic expression vector pGEX- The multiple cloning site region of 4T-2 was used to construct the recombinant expression plasmid pGEX-4T-2-Sjwnt4, and transform and express the host strain BL21(DE3);

⑦Expression in Escherichia coli The identified pGEX-4T-2-Sjwnt4/BL21(DE3) was inoculated in liquid LB medium, cultured with shaking at 37°C, and the expression was induced by adding IPTG with a final concentration of 1mM when the OD value was 0.6. After 0h, 0.5h, 1h, 2h, 4h, and 6h of IPTG induction, the bacteria were collected respectively, and the protein of the bacteria was analyzed by SDS-PAGE to determine the optimal induction time;

⑧Western blotting detection SDS-PAGE electrophoresis was performed on the high-expression phase bacterial protein, transferred to nitrocellulose membrane, and treated with anti-GST monoclonal antibody or pGEX-4T-2/BL21 Escherichia coli lysate respectively. Adult antigen immunized rabbit serum was used as the primary antibody, horseradish peroxidase-labeled goat anti-mouse and goat anti-rabbit IgG were used as the secondary antibody, and DAB was used as the substrate for color development.

Another object of the present invention is to provide the application of Sjwnt-4 coding gene in the preparation of medicines for preventing and treating schistosomiasis.

Cloning and bioinformatics analysis of the SjWnt-4 coding gene of the present invention

In this study, the RACE technology was used to extend the 3' and 5' ends of the EST fragment (GenBank ^TM accession number AAM89872), and then undergo two rounds of PCR amplification respectively. The PCR products were sequenced and spliced to obtain a 2044bp DNA fragment. The coding gene containing the complete reading frame was cloned, and its open reading frame was 1311bp, encoding 436 amino acids. The sequence was searched for homology using NCBI's Blast software, and the results showed no significant homology with other known genes of Schistosoma japonicum. Novel Schistosoma Gene. The results of similarity comparison of amino acid sequences show that it has high homology with Wnt family proteins, among which the homology with Wnt4 protein is the highest, and the analysis of amino acid sequences also shows that it has very typical Wnt family protein characteristics: in the entire protein sequence Scattered with more than 100 conserved sites of Wnt family proteins; rich in cysteine residues that can be cross-linked to form disulfide bonds, about 23 to 24 conserved cysteines, 50% of which are located at the carboxy terminus of the protein ; with three or four glycosylation sites, and selected from Japanese planarian (accession number BAD93239.1), human (GeneBank accession number NP_110388.2), mouse (GeneBank accession number NP_033549.1) , Eugene Kangaroo (GeneBank Accession No. AAY18780.1), Chicken (GeneBank Accession No. JC2451), Nematode (GeneBank Accession No. NP_493668.1), Drosophila (GeneBank Accession No. NP_476810.1) Wnt4 protein for amino acid analysis Multiple alignments of sequences. The results showed that the amino acid sequence encoded by this gene had the highest similarity of 43% (E=1e-100) with the Wnt4 of the planarian japonica belonging to the flatworm phylum, and 37% (E=9e-72) similarity with human Wnt4. ), and the rest are between 36% and 38%. Accordingly, it was speculated that the protein encoded by the gene was Schistosoma japonicum Wnt4 protein, and the gene was named Schistosoma japonicum wnt4 (Sjwnt4) (GeneBank accession number DQ643829).

The results of T cell epitope prediction on the amino acid sequence encoded by the gene showed that the sequence contained multiple antigenic peptides with high binding titer to specific HLA molecules (Table 1).

Table 1 Potential antigenic peptides with higher binding efficiency between Sjwnt4 and HLA

Table1 The peptide binding to HLA with high efficiency in Sjwnt4

Period and sex expression analysis of SjWnt4 gene of the present invention

In order to understand the expression of SjWnt4 gene in different developmental stages of Schistosoma japonicum, the total RNA of 14-day-old larvae, 19-day-old larvae, 31-day-old adults, 44-day-old males and 44-day-old females was extracted, and the housekeeping gene α- Tubulin was used as an internal reference, and the expression of this gene in several different developmental stages of Schistosoma japonicum was detected by fluorescent quantitative PCR method. The experimental results showed that SjWnt4 was expressed in juveniles, adults, and male and female worms of Schistosoma japonicum (Figure 1), and the expression level was highest in juvenile worms at 19 days old, and the expression level in female worms at 44 days was significantly higher than that in male worms.

Construction and Identification of Recombinant Prokaryotic Expression Vector pGEX-4T-2-Sjwnt4

The successful construction of the pGEX-4T-2-Sjwnt4 recombinant expression plasmid was confirmed by PCR, BamHI and Xho I double enzyme digestion (Fig. 2) and sequencing (Fig. 3).

The Sjwnt4 gene of the present invention is expressed in Escherichia coli

The recombinant expression plasmid pGEX-4T-2-Sjwnt4 was expressed in Escherichia coli BL21(DE3), and the results of SDS-PAGE electrophoresis showed that 1mM IPTG induced 4h to reach the maximum expression level; the molecular weight of the recombinant protein was about 76kD, which was consistent with the expected results ( The estimated molecular weight of Sjwnt4 protein is about 49.6kD, and the vector expressed protein GST is about 26kD, so the molecular weight of the recombinant protein is about 75.6kD) (Figure 4). The recombinant protein exists in the form of inclusion body, which is soluble in PBS containing 8M urea.

Antigenicity analysis of the above expression products

Carry out SDS-PAGE electrophoresis with the recombinant expression product, transfer to NC membrane by electrophoresis, use anti-GST monoclonal antibody and pGEX-4T-2/BL21 Escherichia coli protein-adsorbed Schistosoma japonicum adult antigen immune serum as primary antibodies for Western blot respectively , the results all have an obvious recognition band at 75kD (arrow in Figure 5), indicating that the recombinant expression product is a GST fusion protein and has good antigenicity.

The above results showed that the Sjwnt4 gene was expressed in 14-day larvae, 19-day larvae, 31-day worms, 44-day females and 44-day males after S. At other stages; at the same time, the expression level of this gene in 44d females was higher than that in 44d males. It is known from past studies that after Schistosoma japonicum infects the host, the male and female worms congest and pair on the 15th to 16th day, and the yolk gland begins to develop after the male and female worms conjoin, and the yolk cells differentiate to synthesize yolk droplets; on the 22nd day, the female worm expresses egg shell protein; after the 24th day, the ovulation state will be maintained for a long time. The analysis results of the expression status of the above Sjwnt4 gene showed that the expression changes of this gene were closely related to the developmental changes of the sex phenotype of Schistosoma japonicum, revealing from one aspect the important role of Wnt4 gene in the development of the reproductive system of Schistosoma japonicum. This result has some similarities with the biological significance of the expression of this gene in mice and wallabies. In mice, Wnt4 is initially expressed in renal duct mesenchyme and undifferentiated gonads, and it is expressed in undifferentiated gonads of both sexes, but after sex-specific differentiation, Wnt4 expression decreases in male gonads, whereas in male gonads maintained in the female gonad. In marsupials such as the tamar wallaby, Wnt4mRNA is expressed in both undifferentiated gonads of both sexes; the expression level of female wallaby Wnt4mRNA significantly increases during the ovarian differentiation process, reaching the peak after about 9 to 13 days after birth. It then begins to decline after all female germ cells enter meiosis; Wnt4 mRNA expression levels in male wallabies drop immediately after vas deferens formation. Therefore, if human intervention, such as using RNAi interference technology to inhibit the expression of Schistosomiasis Wnt4 or using Wnt4 protein as a drug target to inhibit its activity, can control the sex development and oviposition of Schistosoma japonicum, it will reduce the pathological damage of schistosomiasis and block Communication will be important.

The invention clones and obtains the schistosomiasis Wnt4 gene for the first time, which has great application value for the role of the Wnt signaling pathway in the growth and development of schistosomiasis, especially the development of reproductive organs, and the development of efficient anti-schistosomiasis vaccines and screening of new anti-schistosomiasis drugs.

Nucleotide and deduced amino acid sequences of SjWnt-4.

1 AATATGATGCTACTCCTTATGATGATTCAACATCAGCCAGTAGATTAAACAAAAATAATTACAACACTGAAGGATACCGTCGATCACCAA

91 ATTCTGATTTACGTTTTGTTGAAGCATCAAAAATCACAAGATTTGGATTATCCTCATACAAGTAAAAAATATAAATTCAGACTTAAATA

181 CAAATAATTATAATCATGAACGA

AATCTAACTCAGCTAGAACAAACGATTCAAGACATGAATAATAATGATAGTAACAATATAATGACATCAGAAACATTTGTTGGTGCC204

AATCTAACTCAGCTAGAACAAACGATTCAAGACATGAATAATAATGATAGTAACAATATAATGACATCAGAAACATTTGTTGGTGCC

N D S N N I M T S E T T F V G A M N L T Q L E Q T I Q D M N N

294 GATGGAAAATTACAAATGAGTATATGCGATCATCCAAATGGATTTTTACGGAGACAAAAGAAATTATGCCGTCAGTATTTACATTTGATG

D G K L Q M S I C D H P N G F L R R Q K K K L C R Q Y L H L M

384 GAGAGTGTAATCCGTGGTTATTTTTATGGGTTTAAAAAGAATGTGAATATCAATTTTCTGCACATCGATGGAATTGTCAGGGTCATAACTTA

E S V I R G Y F M G L K E C E Y Q F S A H R W N C Q G H N L

474 ACTATTCAAAGCACCAACTAGTCGAAAACAGAAACGTTTAAGATATAGAGAATCTGAGTTGAAAAATGATATGGATAATTCACGAAGAAAA

T I Q A P T S R K Q K K R L R Y R E S E L K N D M D N S R R K

564 TCTGTACGTATACAAACATACTTAGACAAGCTTTTATCTAAAGGAACAAGAGAATCAGCTTATGTTTTGGCTGTTACATCCGCCGGTGTA

S V R I Q T Y L D K L L S K G T R E S A Y V L A V T S A G V

654 TCACATGCAGTCACTAAAGCATGCAGTAGTGGTCTACATGATAACTGTGGATGTGACAGAACAATATACGATCATCCTAGAGAACCAAAT

S H A V T K A C S S S G L H D N C G C D R T I Y D H P R E P N

744 TTTGAATGGTCAGGATGTTCAGATAATATACATTTTGGAGCAGCATTTTCAAGACAATTTCTTGATGTACGAGAACGTAACAGACTGAAA

F E W S G C S D N I H F G A A F S R Q F L D V R E R N R L K

834 CGTAATCCAAAAATTAGGACTGACAAATTTACATAATAATCATGTGGGAAGACATATGGTAATCAATAAAATGGAAGTCCAGTGCAAATGT

R N P K L G L T N L H N N H V G R H M V I N K M E V Q C K C

924 CATGGTGTAAGTGGTTCATGTGAAATGCGTACATGTTGGCGATCGTTACCGAAATTTCGGCATTTAGGTGCACAATTACAAGAAAGATTT

H G V S G S C E M R T C W R S L P K F R H L G A Q L Q E R F

1014 CATGAAGCAATACAAGTCACTTACATACAAAATCGTCTGGTCTCGATGAAAGCACTAGAACAATTAAGTAAAGAATCAAATGGAAACGCA

  H E A I Q V T Y I Q N R L V S M K A L E Q L S K E S N G N A

1104 CTATTACTTTCTCATTCTGCATTATTATCATCAGTAGTATCAGGAGTATCATCCGATGAATTACCGGCATCACCGCGTATTAATAGA

L L L L S H S A L L L S S V V S G V S S S D E L P A S P R I N R

1194 AATGCGTTTGATACTTTAACTCGTTTCATCATCATTGACTACATCGCCTTTACCATCACCAACTGAAAATGATTTGATTTATATTAGTGAA

N A F D T L T R S T S L T T S P L P S P T E N D L I Y I S E

1284 TCACCAACATTTTGTCATCATGATCCAAGATATGGTAGTATTGGTACATATGGTCGACAGTGTGATGAAAATTCTCAAGGTTTAAACAGT

S P T F C H H D P R Y G S I G T Y G R Q C D E N S Q G L N S

1374 TGTAATTATTTATGCTGTGGTCGAGGATTTAAACGACAAACGTTTGTTCAACAAGAGAGATGTGATTGTAAATTTCAGTGGTGTTGCAAA

C N Y L C C G R G F K R Q T F V Q Q E R C D C K F Q W C C K

TATATATATATATAAGGTATCTTTTTATCGTTCGCAATC1464 GTTGTCTGTAAAACATGTCGTAAAACAGTAGTTATATCTACATGTAAT

TATATATATATATAAGGTATCTTTTTATCGTTCGCAATC

V V V C K T C R K T V V I S T C N *

1554 ACTTTGTGTGTTTTATTTTCATTTTCGTACAATGAACTCCCCTACCCTCTGTAGATCTCTTTCATAACTATTCCAAAAGTTCTTCCTTATTGC

1644 ATTTGTATTACAAAAAGATCTGAATTAAAGTAACAACTCCTGATAATCCTAATCAAAAGCAAAACAAGTTAACTGAGTTTGCAGGGGATGA

1734 AGAAGATTTGAAAACATAAAATTTACTAAGTTGATGACAATTTACTCCAAAGGAAATAAGGAAACGATAAAGAATAACCGAAAGACAA

1824TATATTATTGGTGAATTCACTGTGCTTAAAATAAAAATATCAAGAGAGAACAATCGAATTTGCACACGTTTTTCCTTTCTTCCTAAATTC

1914 TATCTTTTCTATAACACTGTTTCTTGTATATGGTTATTTTCTCAATATACCGATTACATTGTGTATATTCTGTTTACAGTCAATTACTTAT

2004 TAAATCAGTCAGTAACCAAAAAAAAAAAAAAAAAAAAAAA

Description of drawings:

Figure 1: Real-time quantitative RT-PCR detection of the expression of SjWnt4 gene in different stages and sexes of Schistosoma japonicum;

14d: 14-day-old worm; 19d: 19-day-old worm; 31d: body of 31-day worm; 44d(M): 44-day-old male; 44d(F): 44-day-old female.

Figure 2: Double enzyme digestion identification and PCR identification of the recombinant plasmid pGEX-4T-2-Sjwnt4;

M1: DNA Marker DL15 000; 1: pGEX-4T-2-Sjwnt4 recombinant plasmid digested with BamHI and Xho I; 2: pGEX-4T-2 empty vector digested with BamHI and Xho I; M2: DNA marker DL2 000;3: PCR product of pGEX-4T-2-Sjwnt4 recombinant plasmid.

Figure 3: Schematic diagram of pGEX-4T-2-Sjwnt4 recombinant plasmid;

Fig. 5 The recombinant plasma pGEX-4T-2-Sjwnt4.

Figure 4: SDS-PAGE analysis of the expressed proteins of pGEX-4T-2-Sjwnt4/BL21(DE3) in different phases;

M: standard protein molecular weight; 1, 2, 3, 4, 5, 6: expression products of recombinant plasmid pGEX-4T-2-Sjwnt4 after IPTG induction at 0h, 0.5h, 1h, 2h, 4h, 6h; 7: pGEX The product of -4T-2-Sjwnt4 cultured for 6 hours without adding IPTG

Figure 5: Western blot analysis of pGEX-4T-2-Sjwnt4 recombinant protein;

M: Pre-stained standard protein molecular weight; 1: pGEX-4T-2-Sjwnt4 uninduced product; 2: pGEX-4T-2-Sjwnt4 induced expression product; a.: Western blot with mouse anti-GST monoclonal antibody as primary antibody ; b: Western blot using rabbit serum against Schistosoma japonicum adult antigen as the primary antibody.

Detailed ways:

Example 1 Cloning of Schistosoma japonicum signal transduction protein Sjwnt-4 gene

1. Experimental materials

1.1 Materials

Trizol and GeneRacer ^™ Kit were purchased from Invitrogen; Ex Taq Hot Start DNA polymerase and T4 DNA ligase were purchased from TaKaRa Bioengineering (Dalian) Co., Ltd.

1.2 Strains, plasmids and experimental animals

Escherichia coli DH5α was provided by our institute, and the pUCm-T vector was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. New Zealand white rabbits (male, 2.5-3.0 kg) were purchased from Shanghai Luojing Feida Experimental Animal Farm. The cercariae of the mainland China strain of Schistosoma japonicum were provided by the snail laboratory of our institute. New Zealand white rabbits were infected with 20,000, 5,000, and 2,000 cercariae of Schistosoma japonicum by abdominal patch method, and were killed after 14 days, 19 days, 31 days, and 44 days.

2. Method

2.1 Extraction of total RNA Take 200 mg of Schistosoma japonicum 19-day-old larvae frozen in liquid nitrogen, and extract total RNA according to the instructions of the Trizol kit;

2.2 RACE primer design and amplification In our laboratory, Cheng Guofeng and others used two-dimensional electrophoresis combined with peptide mass fingerprinting technology to obtain a peptide sequence of a Wnt family protein, and used this peptide sequence as a query sequence in the Schistosoma japonicum EST library (http:// www.ncbi.nlm.nih.gov/BLAST) found a corresponding EST fragment of Schistosoma japonicum (GenBank ^TM accession number AAM89872), which is 727bp long and does not contain a complete ORF. Using the EST sequence as a template, RACE primers (synthesized by Shanghai Shenneng Bocai Biotechnology Co., Ltd.) were designed and synthesized. Two nested primers for 3′RACE: 3GSP1: 5′-TATGCTGTGGTCGAGGATTTAAACG-3′, 3GSP2: 5′-GGTGTTGCAAAGTTGTCTGTAAAACA-3′; Two nested primers for 5′RACE: 5GSP1: 5′-GGTGTTGCAAAGTTGTCTGTAAAACA-3′, 5GSP2: 5'-TTGTCGTTTAAATCCTCGACCACAG-3'; specific steps were performed according to the operation manual of the GeneRacer ^™ kit. The RACE product was cloned into the pCR4-TOPO vector provided by the kit, and positive clones were selected for sequencing by Handsome Biotechnology Co., Ltd. Use DNAStar software to find the overlap between the 3′ and 5′ RACE product sequences and the original EST sequence, and splice the three sequences;

2.3 Amplification of ORF-containing cDNA fragments Primers (F1 and F2) were designed according to the spliced cDNA sequences to amplify ORF-containing cDNA fragments. F1: 5′-CCG GGATCC ATGAATCTAACTCAGCTAGAA-3′ introduced restriction site BamHI (underlined); F2: 5′-CGG CTCGAG TTAATTACATGTAGATATAAC-3′ introduced restriction site (Xho I) (provided by Shanghai Shenergy Bocai Biology Technology Co., Ltd. Synthesis). Take 3 μl of cDNA obtained by reverse transcription in 3′RACE as a template for PCR amplification. The PCR reaction conditions are 94°C for 5 minutes, followed by 94°C, 30s, 55°C, 30s, 72°C, 1min30s, a total of 30 Cycle, and extend at 72°C for 10 min after the cycle ends. The PCR product was recovered with an agarose gel DNA recovery kit. After purification, it was connected to the pUCm-T vector and transformed into E. coli DH5α competent cells. A single colony was selected for enzyme digestion and identification. The positive plasmid was named pUCm-T-Sjwnt4 and sent to Handsome Biotechnology Limited Sequencing;

2.4 Bioinformatics analysis Perform similarity comparison of the sequenced cDNA on NCBI (http://www.ncbi.nlm.nih.gov/BLAST); use ORF finder on NCBI to find the reading frame of the new gene ; Use DNAstar software to analyze parameters such as the number of amino acid residues, composition, and protein relative molecular weight; use Genetool software to perform multiple comparisons of Wnt proteins in different species; use NetAcet software (http://www.cbs.dtu.dk/services /NetAcet/) for glycosylation site prediction. The MHCII phenotype online prediction tool of SYFPEITHI (http://www.uni-tuebingen.de/uni/kxi/) was used for T cell epitope prediction.

3. Results

The EST fragment (GenBank ^TM accession number is AAM89872) was extended by RACE technology at the 3′ end and 5′ end, respectively, after two rounds of PCR amplification, the PCR product was sequenced and spliced to obtain a 2044bp DNA fragment, which was further cloned by PCR technology The coding gene containing the complete reading frame has an open reading frame of 1311bp and encodes 436 amino acids. The sequence was searched for homology using NCBI's Blast software, and the results showed no significant homology with other known genes of Schistosoma japonicum. Gene. The results of similarity comparison of amino acid sequences show that it has high homology with Wnt family proteins, among which the homology with Wnt4 protein is the highest, and the analysis of amino acid sequences also shows that it has very typical Wnt family protein characteristics: in the entire protein sequence Scattered with more than 100 conserved sites of Wnt family proteins; rich in cysteine residues that can be cross-linked to form disulfide bonds, about 23 to 24 conserved cysteines, 50% of which are located at the carboxy terminus of the protein ; with three or four glycosylation sites, and selected from Japanese planarian (accession number BAD93239.1), human (GeneBank accession number NP_110388.2), mouse (GeneBank accession number NP_033549.1) , Eugene Kangaroo (GeneBank Accession No. AAY18780.1), Chicken (GeneBank Accession No. JC2451), Nematode (GeneBank Accession No. NP_493668.1), Drosophila (GeneBank Accession No. NP_476810.1) Wnt4 protein for amino acid analysis Multiple alignments of sequences. The results showed that the amino acid sequence encoded by the gene had the highest similarity of 43% (E=le-100) with the Wnt4 of the flatworm phylum Wnt4 (E=le-100), and the similarity with human Wnt4 was 37% (E=9e-72 ), and the rest are between 36% and 38%. Accordingly, it was presumed that the protein encoded by the gene was Schistosoma japonicum Wnt4 protein, and the gene was named Schistosoma japonicum wnt4 (Sjwnt4) (GeneBank accession number DQ643829).

Example 2

Expression of Schistosoma japonicum Sjwnt4 Gene in Escherichia coli

1. Experimental materials

1.1 Materials Restriction enzymes BamH I, Xho I, and T4 DNA ligase were purchased from TaKaRa Bioengineering (Dalian) Co., Ltd.

1.2 Bacterial strains and plasmids Plasmids pGEX-4T-2 and BL21(DE3) were provided by our institute.

2. Method

2.1 Construction of recombinant expression plasmid From the recombinant plasmid pUCm-T-Sjwnt4 determined after sequencing, the cDNA fragment of Sjwnt4 containing ORF was excised with restriction endonucleases BamHI and Xho I, and the complete sequence was directional cloned into the prokaryotic expression vector pGEX- The multiple cloning site region of 4T-2 was used to construct the recombinant expression plasmid pGEX-4T-2-Sjwnt4, and transform and express the host strain BL21(DE3);

2.2 Expression in Escherichia coli The identified pGEX-4T-2-Sjwnt4/BL21(DE3) was inoculated in liquid LB medium, cultured with shaking at 37°C, and the expression was induced by adding IPTG at a final concentration of 1 mM when the OD value was 0.6. After 0h, 0.5h, 1h, 2h, 4h, and 6h of IPTG induction, the bacteria were collected respectively, and the protein of the bacteria was analyzed by SDS-PAGE to determine the optimal induction time.

3. Results

The recombinant expression plasmid pGEX-4T-2-Sjwnt4 was expressed in Escherichia coli BL21(DE3), and the results of SDS-PAGE electrophoresis showed that 1mM IPTG induced 4h to reach the maximum expression level; the molecular weight of the recombinant protein was about 76kD, which was consistent with the expected results ( The estimated molecular weight of Sjwnt4 protein is about 49.6kD, and the vector expressed protein GST is about 26kD, so the molecular weight of the recombinant protein is about 75.6kD) (Figure 4). The recombinant protein exists in the form of inclusion body, which is soluble in PBS containing 8M urea.

Claims (2)

1. a Schistosoma japonicum signal transduction protein Sjwnt-4 gene, characterized in that the nucleotide and deduced amino acid sequence of the Sjwnt-4 gene are as follows: 204 AATCTAACTCAGCTAGAACAAACGATTCAAGACATGAATAATAATGATAGTAACAATATAATGACATCAGAAACATTTGTTGGTGCC N D S N N I M T S E T F V G A M N L T Q L E Q T I Q D M N N 294 GATGGAAAATTACAAATGAGTATATGCGATCATCCAAATGGATTTTTACGGAGACAAAAGAAATTATGCCGTCAGTATTTACATTTGATG D G K L Q M S I C D H P N G F L R R Q K K K L C R Q Y L H L M 384 GAGAGTGTAATCCGTGGTTATTTTTATGGGTTTAAAAAGAATGTGAATATCAATTTTCTGCACATCGATGGAATTGTCAGGGTCATAACTTA E S V I R G Y F M G L K E C E Y Q F S A H R W N C Q G H N L 474 ACTATTCAAAGCACCAACTAGTCGAAAACAGAAACGTTTAAGATATAGAGAATCTGAGTTGAAAAATGATATGGATAATTCACGAAGAAAA T I Q A P T S R K Q K K R L R Y R E S E L K N D M D N S R R K 564 TCTGTACGTATACAAACATACTTAGACAAGCTTTTATCTAAAGGAACAAGAGAATCAGCTTATGTTTTGGCTGTTACATCCGCCGGTGTA S V R I Q T Y L D K L L S K G T R E S A Y V L A V T S A G V 654 TCACATGCAGTCACTAAAGCATGCAGTAGTGGTCTACATGATAACTGTGGATGTGACAGAACAATATACGATCATCCTAGAGAACCAAAT S H A V T K A C S S S G L H D N C G C D R T I Y D H P R E P N 744 TTTGAATGGTCAGGATGTTCAGATAATATACATTTTGGAGCAGCATTTTCAAGACAATTTCTTGATGTACGAGAACGTAACAGACTGAAA F E W S G C S D N I H F G A A A F S R Q F L D V R E R N R L K 834 CGTAATCCAAAAATTAGGACTGACAAATTTACATAATAATCATGTGGGAAGACATATGGTAATCAATAAAATGGAAGTCCAGTGCAAATGT R N P K L G L T N L H N N H V G R H M V I N K M E V Q C K C 924 CATGGTGTAAGTGGTTCATGTGAAATGCGTACATGTTGGCGATCGTTACCGAAATTTCGGCATTTAGGTGCACAATTACAAGAAAGATTT H G V S G S C E M R T C W R S L P K F R H L G A Q L Q E R F 1014 CATGAAGCAATACAAGTCACTTACATACAAAATCGTCTGGTCTCGATGAAAGCACTAGAACAATTAAGTAAAGAATCAAATGGAAACGCA   H E A I Q V T Y I Q N R L V S M K A L E Q L S K E S N G N A 1104 CTATTACTTTCTCATTCTGCATTATTATCATCAGTAGTATCAGGAGTATCATCCGATGAATTACCGGCATCACCGCGTATTAATAGA L L L L S H S A L L L S S S V V S G V S S S D E L P A S P R I N R 1194 AATGCGTTTGATACTTTAACTCGTTTCATCATCATTGACTACATCGCCTTTACCATCACCAACTGAAAATGATTTGATTTATATTAGTGAA N A F D T L T R S T S L T T S P L P S P T E N D L I Y I S E 1284 TCACCAACATTTTGTCATCATGATCCAAGATATGGTAGTATTGGTACATATGGTCGACAGTGTGATGAAAATTCTCAAGGTTTAAACAGT S P T F C H H D P R Y G S I G T Y G R Q C D E N S Q G L N S 1374 TGTAATTATTTATGCTGTGGTCGAGGATTTAAACGACAAACGTTTGTTCAACAAGAGAGATGTGATTGTAAATTTCAGTGGTGTTGCAAA C N Y L C C G R G F K R Q T F V Q Q E R C D C K F Q W C C K 1464 GTTGTCTGTAAAACATGTCGTAAAACAGTAGTTATATCTACATGTAAT

V V V C K T C R K T V V I S T C N *. 2. a method for cloning and expression of Schistosoma japonicum signal transduction protein Sjwnt-4 gene as claimed in claim 1, it is characterized in that the method comprises the following steps: (1) Material Trizol, GeneRacer ^TM Kit; Ex Taq Hot Start DNA polymerase, restriction endonuclease BamHI, Xho I, T4 DNA ligase, fluorescent quantitative PCR detection kit and random primers; goat anti-mouse IgG-HRP anti-GST monoclonal antibody; Goat Anti-Rabbit IgG-HRP; Reverse Transcriptase; RNase Inhibitor; SYBR Green I and Calibration; (2) Strains, plasmids and experimental animals Plasmid pGEX-4T-2, Escherichia coli DH5α, BL21 DE3 were provided by Shanghai Veterinary Research Institute; pUCm-T vector; male New Zealand white rabbit, 2.5-3.0kg; Rabbits were infected with 20,000, 5,000, and 2,000 cercariae of Schistosoma japonicum by abdominal patch method, and were killed after 14 days, 19 days, 31 days, and 44 days, and the worms were collected by hepatic portal vein perfusion, and frozen in liquid nitrogen for later use; (3) Method ① Extraction of total RNA Take 200 mg of Schistosoma japonicum 19-day-old larvae frozen in liquid nitrogen, and extract total RNA according to the instructions of the Trizol kit; ② Primer design and amplification for RACE A peptide sequence of a Wnt family protein was obtained by two-dimensional electrophoresis combined with peptide mass fingerprinting technology, and a corresponding EST fragment GenBank ^TM accession number AAM89872 of Schistosoma japonicum was searched in the EST library of Schistosoma japonicum with this peptide sequence as the query sequence, with a length of 727bp. Does not contain a complete ORF, use the EST sequence as a template to design and synthesize RACE primers, two nested primers for 3'RACE: 3GSP1: 5'-TATGCTGTGGTCGAGGATTTAAACG-3', 3GSP2: 5′-GGTGTTGCAAAGTTGTCTGTAAAACA-3′; Two nested primers for 5′ RACE: 5GSP1: 5'-GGTGTTGCAAAGTTGTCTGTAAAACA-3', 5GSP2: 5'-TTGTCGTTTAAATCCTCGACCACAG-3'; The specific steps are carried out according to the operation manual of the GeneRacer ^TM kit. The RACE product is cloned into the pCR4-TOPO vector provided by the kit, the positive clones are selected for sequencing, and the DNAStar software is used to find the overlapping parts of the 3′, 5′ RACE product sequence and the original EST sequence , splicing the three sequences; ③Amplification of cDNA fragments containing ORF Primers F1 and F2 were designed according to the spliced cDNA sequence to amplify the cDNA fragment containing ORF. F1: 5′-CCG GGATCC ATGAATCTAACTCAGCTAGAA-3′ introduced restriction site BamH I and underlined; F2: 5′-CGG CTCGAG TTAATTACATGTAGATATAAC-3 'Introduce the enzyme cutting site Xho I; take 3μl of cDNA obtained by reverse transcription in 3'RACE as a template for PCR amplification. , 72°C, 1min30s, a total of 30 cycles, after the end of the cycle, extend at 72°C for 10min, the PCR product was recovered with an agarose gel DNA recovery kit, purified and connected to the pUCm-T vector, and transformed into Escherichia coli DH5α competent cells, A single colony was selected for enzyme digestion and identification, and the positive plasmid was named pUCm-T-Sjwnt4 and sequenced; ④Bioinformatics analysis The sequenced cDNA was compared on the NCBI for similarity, and the ORF finder on the NCBI was used to find out the reading frame of the new gene; DNAstar software was used to analyze the number of amino acid residues, composition, and protein relative molecular weight; and the clustalW software was used to analyze Multiple comparisons of Wnt proteins from different species; glycosylation site prediction using NetAcet software, and T cell epitope prediction using SYFPEITHI's MHCII phenotype online prediction tool; ⑤Fluorescence real-time quantitative RT-PCR In the experiment, the housekeeping gene α-tubulin of Schistosoma japonicum was selected as the internal reference; the total RNA of 14-day-old and 19-day-old juveniles, 31-day-old adults, 44-day-old males and 44-day-old females were respectively extracted, and cDNA was synthesized with random primers after removing genomic DNA. One strand; fluorescent quantitative PCR primers: primers for amplifying Sjwnt4: S1: 5′-ACATACAAAATCGTCTGGTCTC-3′, S2: 5′-GATGGTAAAGGCGATGTAGTC-3′, the length of the amplified fragment is 214bp; primers for amplifying α-tubulin: T1: 5 ′-CTGATTTTCCATTCGTTTG-3′, T2: 5′-GTTGTCTACCATGAAGGCA-3′, the length of the amplified fragment is 213bp; the primers were synthesized by Shanghai Sangong, and the real-time quantitative PCR was carried out by fluorescent dye method. The reaction system included: 5×R-PCR Buffer 5μl , 250mM Mg ²⁺ 0.3μl, 10mM dNTP 0.75μl, 10μM primer 1.0μl, 25×SYBR Green I 1.0μl, 10-3×Calibration 1.0μl, 5U/μl Ex Taq Hs 0.25μl, ddH ₂ O 14.7μl, template cDNA 1.0 μl, 25 μl in total; reaction parameters: denaturation at 95°C for 3 min, 95°C for 5 s, 58°C for 30 s, 40 cycles, in which the end time point at 58°C for 30 s was the detection point of fluorescence signal, and each reaction was repeated in 3 wells; BIO-RAD's iCycler ^TM IQversion 3.0 software was used for calculation and analysis, and α-tubulin was used as the internal reference to standardize the reaction results to obtain the content of the target gene relative to every million housekeeping genes; ⑥Construction of recombinant expression plasmid From the recombinant plasmid pUCm-T-Sjwnt4 determined after sequencing, use restriction endonucleases BamHI and Xho I to cut out the cDNA fragment of Sjwnt4 containing ORF, and clone the complete sequence into the multi-cloning of prokaryotic expression vector pGEX-4T-2 site region, construct the recombinant expression plasmid pGEX-4T-2-Sjwnt4, and transform and express the host strain BL21DE3; ⑦Expression in Escherichia coli The identified pGEX-4T-2-Sjwnt4/BL21 DE3 was inoculated in liquid LB medium, and cultured with shaking at 37°C. When the OD value was 0.6, IPTG with a final concentration of 1mM was added to induce expression. After IPTG induction, 0h, 0.5h, Collect bacteria at 1h, 2h, 4h, and 6h respectively, and use SDS-PAGE to analyze the protein of the bacteria to determine the optimal induction time; ⑧Western blotting detection SDS-PAGE electrophoresis was performed on the high-expression phase bacterial protein, transferred to nitrocellulose membrane, and then immunized with anti-GST monoclonal antibody or adult Schistosoma japonicum antigen treated with pGEX-4T-2/BL21 E. coli lysate Rabbit serum was used as the primary antibody, horseradish peroxidase-labeled goat anti-mouse and goat anti-rabbit IgG were used as the secondary antibody, and DAB was used as the substrate for color development.

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