Proteins from the webs of nephilengys cruentata, avicularia juruensis and parawixia bistriata spiders isolated from brazilian biodiversity

Abstract

The Present invention relates to molecules isolated from the nucleic acid that encodes spider web proteins or fragments of these or other derivatives of these. The invention also refers to a chimerical gene and an expression vector containing molecules isolated from the nucleic acid that codes for proteins related to the webs of Nephilengys, Cruentata, Avicularia Juruensis and Parawixia Bistriata spiders. Another embodiment of the present invention are transformed cells containing a chimerical gene or an expression vector of the present invention. Yet another embodiment of the present invention relates to a method for obtaining genetically modified organisms containing inventive chimerical genes or expression vectors and a method for obtaining recombinant proteins from the silks of Nephilengys, Cruentata, Aviculana Juruensis and Parawixia Bistriata spiders. Finally, the invention describes products, such as biofilaments and compositions, using the recombinant proteins of the present invention. The discovery of new spider silk proteins, as well as their characterisation and expression in different heterologous systems shall be of great use in numerous areas, such as medicine and industry.

1 . Isolated molecules of spider nucleic acid, comprising:
 a) sequences substantially similar to any of the sequences selected from the group identified as SEQ ID N. 1-19;   b) complements of the sequences described in SEQ ID N. 1-19;   c) reverse complements of the sequences described in SEQ ID N. 1-19;   d) reverse sequences of the sequences described in SEQ ID N. 1-19;   
     
     
         2 . Isolated molecules of spider nucleic acid in accordance with  claim 1 , wherein the sequences are isolated from  Nephilengys cruentata, Avicularia juruensis  and  Parawixia bistriata.    
     
     
         3 . A chimeric gene comprising the isolated molecule of  claim 1 . 
     
     
         4 . A chimeric gene comprising:
 a) a promoter optionally linked to a leader sequence and operationally linked to;   b) a coding sequence substantially similar to any of the sequences set forth in SEQ ID N. 1-19.   
     
     
         5 . A chimeric gene in accordance with  claim 4  wherein said promoter is selected from the group consisting of constitutives, inducibles and tissue-specific. 
     
     
         6 . A chimeric gene in accordance with  claim 5  wherein the tissue-specific promoter is selected from cotton fibre gene promoters. 
     
     
         7 . A chimeric gene in accordance with  claim 6  wherein said cotton fibre gene promoters are selected from the group consisting of E6, H6S, Rac13, LTP, ACP, expansin, CAP, anexine, FbL2A and actin 2. 
     
     
         8 . A chimeric gene in accordance with  claim 3  wherein the promoter contains enhancer elements. 
     
     
         9 . A chimeric gene in accordance with  claim 4  wherein said promoter may be expressed in plants, animals, fungus or insects. 
     
     
         10 . A chimeric gene in accordance with  claim 4  wherein the leader sequence is obtained from the same gene as the promoter used to direct transcription of the isolated nucleic acid molecule. 
     
     
         11 . Expression vector comprising a chimeric gene in accordance with  claim 3 . 
     
     
         12 . An expression vector comprising:
 a) a promoter optionally linked to a leader sequence and operationally linked to;   b) an coding sequence substantially similar to any of the sequences identified as SEQ ID N. 1-19 operationally linked to;   c) a termination signal;   d) an origin of replication;   e) a selective marker; and   f) a cloning site.   
     
     
         13 . Expression vector in accordance with  claim 12  wherein the promoter is selected from the group consisting of constitutives, inducibles and tissue-specific. 
     
     
         14 . An expression vector in accordance with  claim 13  wherein the tissue-specific promoter is selected from cotton fibre gene promoters. 
     
     
         15 . An expression vector in accordance with  claim 14  wherein the cotton fibre gene promoters are selected from the group consisting of E6, H6S, Rac13, LTP, ACP, expansin, CAP, anexin, FbL2A and actin 2. 
     
     
         16 . An expression vector in accordance with  claim 11  wherein the promoter contains enhancer elements. 
     
     
         17 . An expression vector in accordance with  claim 12  wherein said leader sequence is obtained from the same gene as the promoter used to direct transcription of the isolated nucleic acid molecule. 
     
     
         18 . An expression vector in accordance with  claim 12  wherein the promoter may be expressed in plants, animals, fungus or insects. 
     
     
         19 . An expression vector in accordance with  claim 12  wherein the transcription termination signal and the polyadenylation region of the present invention includes, but is not limited to, the SV40 termination signal, the HSV TK adenylation signal, the termination signal of the nopaline synthetase gene (NOS) of  Agrobacterium tumefaciens , the octopine synthetase gene termination signal, the termination signal of the 19S and 35S genes of CaMV, the maize alcohol dehydrogenase gene termination signal, the manopine synthetase gene termination signal, the beta-phaseolin gene termination signal, the ssRUBISCO gene termination signal, the sucrose synthetase gene termination signal, the termination signal of the virus that attacks the  Trifolium subterranean  (SCSV), the termination signal of the trpC gene of  Aspergillus nidulans , and other similar. 
     
     
         20 . An expression vector in accordance with  claim 12  wherein the selective marker is selected from the sequences that confer resistance to antibiotics or visual markers. 
     
     
         21 . An expression vector in accordance with  claim 20  wherein said selective marker is chosen from the gene coding sequences conferring resistance to kanamycin, neomycin, ampicillin, chloranphenicol, streptomycin, hygromycin, geneticin, phosphinotrycin, glyphosate, ammonium gluphosinate, AHAS, BAR and GUS. 
     
     
         22 . Isolated molecules of spider silk protein comprising sequences substantially similar to any one of the sequences selected from the group identified as SEQ ID N. 20-38: 
     
     
         23 . Isolated molecules of spider silk protein in accordance with  claim 22  wherein said sequences are isolated from  Nephilengys cruentata, Avicularia juruensis  and  Parawixia bistriata.    
     
     
         24 . Transformed cell containing a chimeric gene in accordance with  claim 3 . 
     
     
         25 . Transformed cell containing an expression vector in accordance with  claim 11 . 
     
     
         26 . Transformed cell containing an isolated protein molecule in accordance with  claim 22 . 
     
     
         27 . Transformed cell in accordance with  claim 24  wherein the cell originated from any one of the groups consisting of bacteria, fungus, insects, mammals and vegetables. 
     
     
         28 . Plant, or a part thereof, or a propagule or progeny thereof, comprising a chimeric gene in accordance with  claim 3 . 
     
     
         29 . Plant, or a part thereof, or a propagule or progeny thereof, comprising an expression vector in accordance with  claim 11 . 
     
     
         30 . Plant, or a part thereof, or a propagule or progeny thereof, comprising an isolated protein molecule in accordance with  claim 22 . 
     
     
         31 . Plant, or a part thereof, or a propagule or progeny thereof, in accordance with  claim 30  wherein the protein is expressed in seeds. 
     
     
         32 . Animal, or a part thereof, or a progeny thereof, comprising a chimeric gene in accordance with  claim 3 . 
     
     
         33 . Animal, or a part thereof, or a progeny thereof, comprising an expression vector in accordance with  claim 11 . 
     
     
         34 . Animal or a part thereof, or a progeny thereof, comprising an isolated protein molecule in accordance with  claim 22 . 
     
     
         35 . Animal, or a part thereof, or a progeny thereof, in accordance with  claim 34  wherein the protein is expressed in the mammary glands. 
     
     
         36 . Microorganism, or a part thereof, comprising a chimeric gene in accordance with  claim 3 . 
     
     
         37 . Microorganism, or a part thereof, comprising an expression vector in accordance with  claim 11 . 
     
     
         38 . Method for producing a genetically modified organism comprising the following stages:
 a) transforming a cell, tissue, organ or embryo with a chimeric gene in accordance with  claim 3  or an expression vector in accordance with  claim 11 ;   b) selecting transformed cells, cell calluses, embryos or seeds;   c) regenerating mature plants, mature embryos or microorganisms of the transformed cells, cell calluses, embryos or seeds selected in stage (b);   d) selecting mature plants, mature embryos or microorganisms cells of stage (c) containing the chimeric gene or expression vector with the nucleotide sequences that encode the spider silk protein.   
     
     
         39 . Method for the production of recombinant protein comprising the following stages:
 a) transforming a cell, tissue, organ or embryo with an expression vector in accordance with  claim 11 ;   b) selecting transformed cells, cell calluses, embryos or seeds;   c) regenerating mature plants, mature embryos or microorganisms of the transformed cells, cell calluses, embryos or seeds selected in stage (b);   d) selecting mature plants, mature embryos or microorganisms cells of stage (c) containing the expression vector with the nucleotide sequences that encode the spider silk protein.   e) extracting of the recombinant spider silk protein produced in the organisms selected in stage (d).   
     
     
         40 . Recombinant protein obtained by the method described in  claim 39 . 
     
     
         41 . Recombinant protein in accordance with  claim 40  characterised by the fact the protein presents microbicide activity. 
     
     
         42 . Recombinant protein in accordance with  claim 41  wherein the microbicide activity is against viral replication. 
     
     
         43 . Recombinant protein in accordance with  claim 40  wherein said protein presents defensin activity. 
     
     
         44 . Recombinant protein in accordance with  claim 43  wherein said defensin activity is against pests and insects. 
     
     
         45 . Recombinant protein in accordance with  claim 40  wherein said the protein presents dermatological activity. 
     
     
         46 . Dermatological composition comprising:
 a) A recombinant protein in accordance with  claim 40 ;   b) a pharmaceutically acceptable vehicle.   
     
     
         47 . Dermatological composition in accordance with  claim 46  wherein the pharmaceutically acceptable vehicle is selected from the group consisting of glycerol, water, saline solution, ethane, solutions with phosphates and organic acid salts. 
     
     
         48 . Microbicide composition comprising:
 a) A recombinant protein in accordance with  claim 41 ;   b) an agriculturally acceptable vehicle and, optionally,   c) additives.   
     
     
         49 . Microbicide composition in accordance with  claim 48  wherein the agriculturally acceptable vehicle is selected from the group consisting of water, organic solvents, humectants, preservatives, thickeners, antimicrobial agents, antioxidants, emulsifiers, film forming polymers and mixtures of these. 
     
     
         50 . Microbicide composition in accordance with  claim 48  wherein the additives are selected from the group consisting of rosin gum, latex, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyethylene, polyvinyl acetate and mixtures of these. Further optional additives include methyl, methacrylate and mixtures of these. 
     
     
         51 . Biopolymers produced from the recombinant protein obtained in accordance with the method described in  claim 39 .