Clone, expression and use of Schistosoma Japonicum signal transduction protein Sjwnt-4 gene
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A signal transduction and schistosome technology, applied in the field of genes, can solve the problems of slow progress, no research report on the Wnt gene of schistosomes, and little research on the signal transduction system of schistosome growth and development.
Inactive Publication Date: 2010-10-13
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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Problems solved by technology
But so far, little research has been done on the signal transduction system of schistosome growth and development, resulting in slow progress in the development of highly effective candidate vaccine molecules and the screening of new drugs
However, there are no research reports on the Wnt gene of schistosomiasis at home and abroad.
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Embodiment 1
[0090] Example 1 Cloning of Schistosoma japonicum signal transduction protein Sjwnt-4 gene
[0091] 1. Experimental materials
[0092] 1.1 Materials
[0093] Trizol, Gene Racer TM Kit was purchased from Invitrogen; Ex Taq Hot Start DNA polymerase and T4 DNA ligase were purchased from TaKaRa Bioengineering (Dalian) Co., Ltd.
[0094] 1.2 Strains, plasmids and experimental animals
[0095] 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.
[0096] 2. Meth...
Embodiment 2
[0104] Expression of Schistosoma japonicum Sjwnt4 Gene in Escherichia coli
[0105] 1. Experimental materials
[0106] 1.1 Materials Restriction enzymes BamH I, Xho I, and T4 DNA ligase were purchased from TaKaRa Bioengineering (Dalian) Co., Ltd.
[0107] 1.2 Bacterial strains and plasmids Plasmids pGEX-4T-2 and BL21(DE3) were provided by our institute.
[0108] 2. Method
[0109] 2.1 Construction of recombinant expression plasmid From the recombinant plasmid pUCm-T-Sjwnt4 determined after sequencing, the cDNA fragment containing ORF of Sjwnt4 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);
[0110] 2.2 Expression in Escherichia coli The identified pGEX-4T-2-Sjwnt4 / BL21(DE3) was inoculated in liquid LB m...
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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 indicates that: 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 gene 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 gene is 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 schistosomulum, 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 colisystem 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
Technical field: [0001] 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: [0002] 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 der...
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