BIFUNCTIONAL FUSION PROTEINS TO INHIBIT ANGIOGENESIS IN TUMOR MICROENVIRONMENT AND TO ACTIVATE ADAPTIVE IMMUNE RESPONSES AND THE GENES AND USES THEREOF

Abstract

Bifunctional fusion proteins having Tumstatin active fragments and CD137L extracellular regions are provided. The proteins exhibit activities to inhibit the proliferation of human umbilical vein endothelial cells and to costimulate the proliferation of T cells. They can be used for the treatment of various tumor-related diseases and the regulation of angiogenesis and immunological effects in humans.

Claims

1. A bifunctional recombinant protein possessing the activities of Tumstatin and CD137L, wherein the protein comprises amino acid sequences of the active fragments of Tumstatin and the CD137L extracellular region, wherein the sequences of Tumstatin and CD137L are fused together through a flexible peptide linker, wherein the amino acid sequence of Tumstatin active fragment is selected from SEQ ID NO:65 to SEQ ID NO:68, wherein the amino acid sequence of the peptide linker is selected from SEQ ID NO:69 to SEQ ID NO:76, and wherein the amino acid sequence of CD137L extracellular region is selected from SEQ ID NO:77 to SEQ ID NO:80.

2. A bifunctional recombinant protein according to claim 1, wherein the protein contains the amino acid sequence selected from SEQ ID NO:25 to SEQ ID NO:48.

3. A gene encoding the bifunctional recombinant protein according to claim 1, wherein the gene comprises the gene encoding Tumstatin active fragment, peptide linker and CD137L extracellular region, wherein the gene encoding the active fragment of Tumstatin is selected from SEQ ID NO:49 to SEQ ID NO:52, wherein the gene encoding peptide linker is selected from SEQ ID NO:53 to SEQ ID NO:60, and wherein the gene encoding CD137L extracellular region is selected from SEQ ID NO:61 to SEQ ID NO:64.

4. A gene encoding the bifunctional recombinant protein according to claim 1, wherein the gene contains a nucleotide sequence selected from SEQ ID NO:1 to SEQ ID NO:24.

5. A gene encoding the bifunctional recombinant protein possessing the activities of Tumstatin and CD137L, wherein it has a 70% or more homology compared with the nucleotide sequence according to claim 3.

6. A preparation method for the bifunctional recombinant protein possessing the activities of Tumstatin and CD137L according to claim 1, wherein the method includes the following steps: (1) design and obtain a nucleotide sequence wherein the gene comprises the gene encoding Tumstatin active fragment, peptide linker and CD137L extracellular region, wherein the gene encoding the active fragment of Tumstatin is selected from SEQ ID NO:49 to SEQ ID NO:52, wherein the gene encoding peptide linker is selected from SEQ ID NO:53 to SEQ ID NO:60, and wherein the gene encoding CD137L extracellular region is selected from SEQ ID NO:61 to SEQ ID NO:64; (2) construct a expression system containing the nucleotide sequence, including constructing the expression vector and transforming the expression vector into a host cell to form a recombinant cell that can express the bifunctional recombinant protein possessing the activities of Tumstatin and CD137L; (3) culture the recombinant cell obtained from step (2); (4) isolate and purify the bifunctional recombinant protein possessing the activities of Tumstatin and CD137L.

7. A preparation method according to claim 6, wherein the expression system is a prokaryotic expression system or a eukaryotic expression system, wherein the prokaryotic expression system is selected from Escherichia coli or Bacillus subtilis, wherein eukaryotic expression system is selected from yeast.

8. The application of the bifunctional recombinant protein possessing the activities of Tumstatin and CD137L according to claim 1, wherein the bifunctional recombinant protein can be applied in preparing pharmaceutical agents for inhibiting angiogenesis in tumor microenvironment, treating various tumor-related diseases, regulating body immunity, stimulating T cell proliferation and synthesizing and secreting cytokines.

Description

BRIEF DESCRIPTION

[0062] Some of the embodiments will be described in detail, with reference to the following figures, wherein like designations denote like members, wherein:

[0063] FIG. 1 is a schematic representation showing the structures of the recombinant proteins possessing the activities of Tumstatin and CD137L described in embodiments of the present invention. The amino acid sequence of the flexible peptide linker is selected from anyone of the group from SEQ ID NO:69 to SEQ ID NO:76.

[0064] FIG. 2 is a schematic view illustrating the location of the amino acid sequences of CD137L1 (SEQ ID NO:77), CD137L5 (SEQ ID NO:78), CD137L6 (SEQ ID NO:79) and CD137L4 (SEQ ID NO:80) in the full-length of CD137L.

[0065] FIG. 3 shows the expression of the recombinant proteins Tumstatin-Peptide linker-CD137L in E. coli, the amino acid sequences of which are from SEQ ID NO:25 to SEQ ID NO:48. Lanes 1-24 correspond to SEQ ID NO:25-48, respectively.

[0066] FIG. 4 shows the representative expression of the recombinant proteins Tumstatin-Peptide linker-CD137L (SEQ ID NO:29 to SEQ ID NO:38) in Bacillus subtilis. Lanes 1-8 correspond to SEQ ID NO:29, 30, 31, 32, 33, 34, 35, 36, 37 and 38 respectively.

[0067] FIG. 5 shows the representative expression of the recombinant proteins Tumstatin-Peptide linker-CD137L (SEQ ID NO:29 to SEQ ID NO:36) in Pichia pastoris. Lanes 1-16 correspond to SEQ ID NO:29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42, respectively.

[0068] FIG. 6 is the SDS-PAGE of the representative renatured samples of Tumstatin-Peptide linker-CD137L by dilution. Wherein, the amino acid sequences of Lane 1-6 correspond to SEQ ID NO:25, 26, 27, 37, 38 and 41, respectively; the amino acid sequences of Lane 7 and 10 correspond to SEQ ID NO:43; the amino acid sequences of Lane 8 and 11 correspond to SEQ ID NO:44; the amino acid sequences of Lane 9 and 12 correspond to SEQ ID NO:48; the amino acid sequences of Lane 13 and 15 correspond to SEQ ID NO:45; the amino acid sequences of Lane 14 and 16 correspond to SEQ ID NO:46; the amino acid sequences of Lane 17 and 18 correspond to SEQ ID NO:47.

[0069] FIG. 7 shows the effects of the representative samples of Tumstatin-Peptide linker-CD137L on the proliferation of human umbilical vein endothelial cells.

[0070] FIG. 8 shows the effects of the representative samples of Tumstatin-Peptide linker-CD137L on the proliferation of mouse T lymphocytes.

DETAILED DESCRIPTION

[0071] Hereinbelow, embodiments of the invention will be further explained in more detail by way of the following examples and drawings.

[0072] Embodiments of the present invention provide 24 types of bifunctional recombinant proteins possessing activities of Tumstatin and CD137L. The nucleotide sequences of these proteins are shown from SEQ ID NO:1 to SEQ ID NO:24, and the amino acid sequences are shown from SEQ ID NO:25 to SEQ ID NO:48. This group of proteins is made up of one Tumstatin active fragment (Tumstatin1 contains amino acid residues from 45 to 98, Tumstatin2 contains amino acid residues from 60 to 132, Tumstatin3 contains amino acid residues from 60 to 98, Tumstatin7 contains amino acid residues from 74 to 98), one CD137L extracellular domain (CD137L1 contains amino acid residues from 46 to 254, CD137L4 contains amino acid residues from 50 to 240, CD137L5 contains amino acid residues from 83 to 254, CD137L6, a combined fragment, contains amino acid residues from 46 to 85 and from 167 to 254), and a peptide linker with anyone of the amino acid sequences from SEQ ID NO:69 to SEQ ID NO:76. In embodiments of the present invention, two restriction enzyme sites consisting of 5-EcoRI and 3-BamHI are designed in the nucleotide sequences (shown from SEQ ID NO:81 to SEQ ID NO:94) containing the the active fragment of Tumstatin and the peptide linker described above. The sequences were synthesized and ligated with plasmid pBluescriptII SK (+) containing the same restriction sites by Shanghai Generay Biological Engineering CO. Ltd.

[0073] All the other expression vectors were purchased from Novagen, and E. coli strain Top10 and BL21 (DE3) were from Invitrogen. Vector pMD18-T, solution I (# D103A), reverse transcriptase, T4 DNA ligase, restriction endonuclease, such as, NdeI and NheI, were obtained from TAKARA Bio. The synthesis of primers and nucleotide sequencing were completed by Shanghai Invitrogen Biotechnology Company. The urea (AR) used for denaturation and dilution refolding was purchased from Nanjing Chemical Reagent Co., Ltd. Human umbilical vein endothelial cells were purchased from Nanjing KGI Biological Technology Development Co., Ltd. EasySep Negative Selection Kit used for mouse T cell proliferation assay was purchased from Stem Cell Company. Anti-CD3 and anti-CD28 monoclonal antibody were purchased from Santa Cruz Company. Other reagents were of analytical grade made in China.

Example 1: The Expression of the Recombinant Protein Tumstatin-CD137L Possessing Activities of Tumstatin and CD137L in E. coli

[0074] 1. Construction of the Expression Systems

[0075] Embodiments of the present invention provide 24 types of bifunctional recombinant proteins possessing activities of Tumstatin and CD137L, which is a fusion of Tumstatin1/Tumstatin2/Tumstatin3/Tumstatin7 and CD137L1/CD137L4/CD137L5/CD137L6 through the combination of a peptide linker (FIG. 1). Wherein, CD137L1, CD137L4, CD137L5 and CD137L6 are the amino acid residues 46-254, 50-240, 83-254, 46-85 plus 167-254 of the full-length of CD137L (FIG. 2), respectively. The nucleotide sequences of the protein are shown from SEQ ID NO:1 to SEQ ID NO:24, the amino acid sequences are shown from SEQ ID NO:25 to SEQ ID NO:48, and the peptide linker is selected from one of the sequences from SEQ ID NO:69 to SEQ ID NO:76.

[0076] The nucleotide sequences encoding the amino acid sequences of CD137 extracellular domain (CD137L1 contains amino acid residues from 46 to 254, CD137L4 contains amino acid residues from 50 to 240, CD137L5 contains amino acid residues from 83 to 254, CD137L6, a combined fragment, contains amino acid residues from 46 to 85 and from 167 to 254) are shown from SEQ ID NO:61 to SEQ ID NO:64, and obtained from PCR amplification. Wherein, the full nucleotide sequence of CD137L could be obtained through the published method (Wang shuzhen. J Ind Microbiol Biotechnol. 2012 March; 39(3):471-6. Doi: 10.1007/s10295-011-1045-1.).

[0077] Preparation of CD137L template used in embodiments of the present invention by employing the published method described above. Using the upstream primer of SEQ ID NO:61 (SEQ ID NO:95) and the downstream primer of SEQ ID NO:61 (SEQ ID NO:96) as primers, CD137L1 (SEQ ID NO: 61) (with two restriction sites consisting of 5-BamHI and 3-NotI) was obtained by PCR amplification with rTaq DNA polymerase and ligated with plasmid pMD18-T. By employing the traditional molecular biology methods (such as enzyme digestion and ligation), the CD137L1 was then cut from plasmid pMD18-T and subcloned into plasmid pBluescriptII SK (+), which contains the gene of Tumstatin and peptide linker (SEQ ID NO:87-SEQ ID NO:94) and was synthesized by Shanghai Generay BiologicalEngineering CO. Ltd. Then, the whole nucleotide sequences of the fusion protein (SEQ ID NO:5-SEQ ID NO:12) were cloned into plasmid pBluescriptII SK(+). Using SEQ ID NO:97 as upstream primer and SEQ ID NO:98 as downstream primer, the genes of the fusion proteins with two restriction sites consisting of 5-NheI and 3-NdeI were amplified by PCR and subcloned into the expression vector pET-11a.

[0078] Preparation of the CD137L template used in embodiments of the present invention by employing the published method described above. Using the upstream primers of SEQ ID NO:62 (SEQ ID NO:99) and the downstream primer of SEQ ID NO:62 (SEQ ID NO:100) as primers, CD137L5 (SEQ ID NO: 62) (with two restriction sites consisting of 5-BamHI and 3-NotI) was obtained by PCR amplification with rTaq DNA polymerase and ligated with plasmid pMD18-T. By employing the traditional molecular biology methods (such asenzyme digestion and ligation), the CD137L5 was then cut from plasmid pMD18-T and subcloned into plasmid pBluescriptII SK (+), which contains the genes of Tumstatin and peptide linker (SEQ ID NO:81-SEQ ID NO:96) and were synthesized by Shanghai Generay BiologicalEngineering CO. Ltd. Then, the whole nucleotide sequences of the fusion proteins (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:13-SEQ ID NO:16) were cloned into plasmid pBluescriptII SK (+). Using SEQ ID NO:101-104 as upstream primers and SEQ ID NO:105 as downstream primers, the genes of the fusion proteins with two restriction sites consisting of 5-NheI and 3-NdeI was amplified by PCR and subcloned into the expression vector pET-11a.

[0079] Preparation of the CD137L template used in embodiments of the present invention by employing the published method described above. Using the upstream primer 1 of SEQ ID NO:63 (SEQ ID NO:106) and the downstream primer 1 of SEQ ID NO:63 (SEQ ID NO:107), the upstream primer 2 of SEQ ID NO:63 (SEQ ID NO:108) and the downstream primer 2 of SEQ ID NO:63 (SEQ ID NO:109) as primers, two fragments of CD137L6 (SEQ ID NO: 63) were obtained by PCR amplification with rTaq DNA polymerase and recovered from the agarose gel. Employing these two fragments as templates, the upstream primer 1 of CD137L6 (SEQ ID NO: 107) and downstream primer 2 of CD137L6 (SEQ ID NO: 109) as primers, CD137L6 (with two restriction sites consisting of 5-BamHI and 3-NotI) was obtained through OverlapPCR amplification and ligated with plasmid pMD18-T. The CD137L6 (SEQ ID NO: 63) was then cut from plasmid pMD18-T and subcloned into plasmid pBluescriptII SK (+), which contains the genes of Tumstatin and peptide linker (SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:84 and SEQ ID NO:86) and were synthesized by Shanghai Generay Biological Engineering CO. Ltd. Using SEQ ID NO:112-113 as primer, the genes of the fusion proteins (SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:17 and SEQ ID NO:18) were obtained by PCR amplification and subcloned into the expression vector pET-11a.

[0080] All the primers involved in the experimental procedures described above are listed as follows:

TABLE-US-00001 UpstreamprimerofSEQIDNO:61 5-GGATCCGCCGTCCTTCTCGCCTGCC-3 (BamHIrestrictionsiteisunderlined) DownstreamprimerofSEQIDNO:61 5-GCGGCCGCTTCCGACCTCGGTGAAGGGAGT-3 (NotIrestrictionsiteisunderlined) Upstreamprimerofthefusionprotein gene(SEQIDNO:5-SEQIDNO:12) 5-GCTAGCACAATGCCATTCTTATTCTGCAATG-3 (NheIrestrictionsiteisunderlined) Downstreamprimerofthefusionprotein gene(SEQIDNO:5-SEQIDNO:12) 5-CATATGTTCCGACCTCGGTGAAGGGAGTCCG-3 (NdeIrestrictionsiteisunderlined) UpstreamprimerofSEQIDNO:62 5-CGGGATCCGCCTCTTGGACCTGCGCGGCAG-3 (BamHIrestrictionsiteisunderlined) DownstreamprimerofSEQIDNO:62 5-GCGGCCGCTTCCGACCTCGGTGAAGGGAG-3 (NotIrestrictionsiteisunderlined) UpstreamprimerofSEQIDNO:1 5-GCTAGCGGTTTTTCTTTCTATTTGTTCAAG-3 (NheIrestrictionsiteisunderlined) UpstreamprimerofSEQIDNO:3 5-GCTAGCGGTTTTTCTTTCTTATTTGTTCAAG-3 (NheIrestrictionsiteisunderlined) UpstreamprimerofSEQIDNO:13-SEQIDNO:14 5-GCTAGCCAAGATTTAGGTACTTTGGGCTCTT-3 (NheIrestrictionsiteisunderlined) UpstreamprimerofSEQIDNO:15-SEQIDNO:16 5-GCTAGCAAGAGCCCAAAGTACCTAAATCTTG-3 (NheIrestrictionsiteisunderlined) DownstreamprimerofSEQIDNO:1,SEQIDNO:3, and SEQIDNO:13-SEQIDNO:16 5-CATATGTTCCGACCTCGGTGAAGGAG-3 (NdeIrestrictionsiteisunderlined) Upstreamprimer1ofSEQIDNO:63 5-CGGGATCCGCCGTCTTCCTCGCCTGC-3 (BamHIrestrictionsiteisunderlined) Downstreamprimer1ofSEQIDNO:63 5-CGCAAACATGCCCTGCCCTG-3 Upstreamprimer2ofSEQIDNO:63 5-CAGGGCAGGGCATGTTTGCGGGTTTCCAGGGCCGCTTGC-3 Downstreamprimer2ofSEQIDNO:63 5-GCGGCCGCTTCCGACCTCGGTGAAGGGAG-3 (NotIrestrictionsiteisunderlined) UpstreamprimerofSEQIDNO:2andSEQIDNO:4 5-GCTAGCGGTTTTTCTTTCTTATTTGTTCAAG-3 (NheIrestrictionsiteisunderlined) UpstreamprimerofSEQIDNO:17 5-GCTAGCCAAGATTTAGGTACTTTG-3 (NheIrestrictionsiteisunderlined) UpstreamprimerofSEQIDNO:18 5-GCTAGCCAAGATTTAGGTACTTTGGGCTCTT-3 (NheIrestrictionsiteisunderlined) DownstreamprimerofSEQIDNO:2,SEQIDNO:4, SEQIDNO:17andSEQIDNO:18 5-CATAGTTCCGACCTCGGTGAAGGGAG-3 (NdeIrestrictionsiteisunderlined)

[0081] PCR amplifications were performed using the following conditions:

[0082] 94 C. 5 min

[0083] 94 C. 1 min, 60 C. 30 s, 72 C. 30 s, 30 cycles

[0084] 72 C. 5 min

[0085] 4 C., Hold

[0086] After being recovered from the gel, the PCR products were ligated with plasmid pMD-18T in a total volume of 20 L containing 1 L of pMD-18T, 4 L of target gene and 15 L of solution. The ligation was carried out at 16 C. for 16 h. The ligation mixture was then used to transform E. coli strain Top10 by calcium chloride method. After being cultured in the solid LB culture medium with ampicillin for 12 h, single clones were picked. The extracted plasmids were then verified by double enzyme digestion with NdeI and NheI. To determine the correctness of the gene sequences, the colonies were sequenced by Shanghai Invitrogen Biotechnology Co. The target genes with expected sequences were ligated with the prokaryotic expression vector pET-11a, wherein the reaction system contains 1 L of T4 ligase 10 buffer, 1 L of pET-11a, 7 L of target gene and 1 L of T4 ligase, and the ligation was carried out at 16 C. for 16 h. After verified by double enzyme digestion, the gene was transformed into the expression system E. coli BL21 (DE3).

[0087] 2. The Expression of the Recombinant Proteins

[0088] 1 mL of E. coli BL21 (DE3) carrying a positive recombinant plasmid was inoculated to 100 mL of LB medium containing 100 g/mL ampicillin under a super clean bench and cultured at 37 C. in incubator shaker set 250 rpm overnight. The activated seed broth was transferred into sterilized LB medium, in which 100 M of IPTG was added at an OD.sub.600 of 0.9. Cells were then harvested by centrifugation (12000 rpm, 30 min) under room temperature after culturing for 8 h at 37 C. Samples (1 g) were re-suspended in 10 mL of PBS, and lysed by high-pressure cell-disruption systems. The lysate was centrifuged for 30 min at 12,000 rpm at 4 C., and the supernatant was discarded and the precipitate was analyzed with SDS-PAGE.

[0089] The procedure of SDS-PAGE is described as follows:

[0090] 1) Wash the appropriate amount of the precipitate with 1 mL of ddH2O, and centrifuge at 12000 rpm for 1 min. After discarding the supernatant, the precipitate was resuspended with 100 L of ddH2O.

[0091] 2) Mix 5 L of 5SDS-PAGE loading buffer with 20 L of the resuspended samples and heat them in boiling water for 5 min.

[0092] 3) Make the 5% stacking gel, 12% separating gel and 1Tri-Gly running buffer according to the Molecular Cloning: A Laboratory Manual.

[0093] 4) Load 20 L of the prepared samples into wells. Run the electrophoresis at a 90V for 30 min and another 130V for 1 h. Remove SDS-PAGE gel, and use coomassie blue to stain for 1-2 h. The gel is then washed three 3 times with ddH2O, and placed in the bleaching solution overnight.

[0094] 5) The results of the protein electrophoresis are shown in supplemented drawings. The expression of the target proteins was significantly increased in the positive bacteria as compared to negative control.

[0095] Detailed expressions of the target proteins are shown in FIG. 3. It can be observed that all proteins described above were expressed in E. coli BL (DE3).

[0096] 3. Purification of the Recombinant Proteins

[0097] Investigation on the Renaturation of Inclusion Bodies

[0098] The bacteria cells were crushed by high-pressure cell-disruption systems as described above. The precipitates were collected and washed twice with washing solution A and B by centrifugation at 12000 rpm for 10 min each time. Solution A consists of 20 mM Tris-HCL, pH8.5, 2 M urea and 2% TritonX-100. Solution B consists of 20 mM Tris-HCL, pH8.5, 2 M urea and 5 mM EDTA. After washing, the precipitates were solubilized at 50 C. overnight with a dissolving solution (20 mM Tris, pH8.5, 8 M urea). After it was completely denatured, the denaturing solution was added to the refolding solution via a constant flow apparatus in chromatography cabinet at 4 C., the solution was stirred at the same time and the process lasted for 12 h. The refolding solution consists of 20 mM Tris-HCL, pH8.5, 2 M urea, 0.4-0.6 M L-Arg and 1 mM EDTA. The solution described above was subjected to ultrafiltration at 4500 rpm for 30 min using Amico Ultra-15 concentration units (Millpore) to concentrate to 1 mL at 4 C. At the same time, the solution was substituted into PBS (pH7.4, 100 mM). The product was preserved at 4 C. (FIG. 6). It was calculated that the purity of the target products after renaturation was greater than 80%, indicating that the denatured inclusion bodies described above were refolded well.

Example 2: The Expression of the Recombinant Proteins Tumstatin-CD137L Possessing Activities of Tumstatin and CD137L in Bacillus subtilis

[0099] 1. Construction of the Expression System

[0100] Using the plasmid pBluescriptII SK (+) harboring the complete gene sequences (SEQ ID NO:1-24) of the recombinant proteins constructed in Example 1 as template, PCR amplification of the different fusion genes with different primers with PstI and HindIII at 5 and 3 termini, respectively. After being double enzyme digestion, the PCR products were subcloned into pasmid pP43. The experimental procedure for PCR, enzyme digestion and ligation were the same to those described in Example 1. The constructed plasmid was then transformed into Bacillus subtilis WB800 by electroporation (detailed methods can be found in the operating manual of electroporation instrument, Bio-Rad).

[0101] 2. Expression of the Recombinant Protein

[0102] Seed broth culturing: 10 L of Bacillus subtilis strain WB800 harboring the plasimid containing target gene was incubated into 5 mL liquid LB medium containing 50 g/mL kanamycinunder a super clean bench and cultured at 37 C. on a rotary shaker at 250 rpm for 12 h. Fermentation of the engineered WB800: the activated seed broth was transferred to the aseptic 2YT medium containing 50 g/mL kanamycin with an inoculation volume of 10% and cultured at 37 C. for 96 h, pH7.0. The cells were collected by centrifugation at 4 C. and analyzed by SDS-PAGE.

[0103] 3. Purification of the Recombinant Proteins

[0104] The recombinant proteins were purified by DEAE-sephadex anion exchange chromatography (GE) with PBS, pH8.5.

Example 3: The Expression of the Recombinant Proteins Tumstatin-CD137L Possessing Activities of Tumstatin and CD137L in Pichia pastoris

[0105] 1. Construction of the Expression System

[0106] The genes (SEQ ID NO:1-24) of recombinant proteins were obtained by double enzyme digestion of the plasmid pPIC9K with EcoRI and NotI and ligated with plasmid pPIC9K. The constructed plasmid was then transformed into Pichia pastoris strain GS115 by electroporation (detailed methods can be found in the operating manual of electroporation instrument, Bio-Rad).

[0107] 2. Expression of the Recombinant Proteins

[0108] P. pastoris was cultivated firstly in rich BMGY medium and then BMMY medium for induction of target protein expression. On the first day, the engineered strains constructed above were inoculated into YPD seed medium, and cultured at 37 C. overnight. On the second day, appropriate amount of seed broth was inoculate to BMGY medium and cultured for 48 h. When the value of OD.sub.600 reached 10, the medium was replaced with BMMY medium. The strains were grown in BMMY medium for 96 h. The cells were collected by centrifugation at 12,000 rpm for 30 min and analyzed by SDS-PAGE.

[0109] 3. Purification of Recombinant Proteins

[0110] The recombinant proteins were purified by DEAE-sephadex anion chromatography (GE) with PBS, pH8.5. The purity of products was greater than 80%.

Example 4: Effects of the Bifunctional Recombinant Proteins Possessing the Activities of Tumstatin and CD137L on the Proliferation of Human Umbilical Vein Endothelial Cells

[0111] 1) Cell culture: The human umbilical vein endothelial cells (HUVEC) were cultured at 37 C. under constant saturated humidity with 5% CO.sub.2 in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 g/mL of streptomycins for 23 d. The cells under exponential-phase growth were used for the following experiments.

[0112] 2) MTT assay: The cells were seeded in 96-well plates at 5000 cells per well by adding 100 L cell suspension to each well and cultured in cell culture incubator for 12 h. Then 100 L of protein samples with different concentrations were added. HUVECs treated with TNP-470 were used as positive control and treated with PBS were used as negative controls. The cells were cultured in triplicate for 48 h. After initiating the cultures for 44 h, 20 L MTT (5 mg/mL) was added. The medium was then softly removed at 48 h and 150 L of DMSO was added into each well. The blue-purple precipitates were completely dissolved after shaking the 96-well plate for 10 min. Then the absorbance of each well at 490 nm was measured by a microplate reader.

[0113] The results are shown in FIG. 7, wherein, TNP-470 is the positive control, 115 is Tumstatin1-Peptide linker 1-CD137L5 (SEQ ID NO:25), 155 is Tumstatin1-Peptide linker 5-CD137L 5 (SEQ ID NO:27), 116 is Tumstatin1-Peptide linker 1-CD137L6 (SEQ ID NO:26), 215 is Tumstatin2-Peptide linker 1-CD137L5 (SEQ ID NO:37), 255 is Tumstatin2-Peptide linker 5-CD137L5 (SEQ ID NO:38), 256 is Tumstatin2-Peptide linker 5-CD137L 6 (SEQ ID NO:41), 711 is Tumstatin7-Peptide linker 1-CD137L1 (SEQ ID NO:29), 721 is Tumstatin7-Peptide linker 2-CD137L1 (SEQ ID NO:30), 731 is Tumstatin7-Peptide linker 3-CD137L1 (SEQ ID NO:31), 741 is Tumstatin7-Peptide linker 4-CD137L1 (SEQ ID NO:32), 751 is Tumstatin7-Peptide linker 5-CD137L1 (SEQ ID NO:33), 761 is Tumstatin7-Peptide linker 6-CD137L1 (SEQ ID NO:34), 771 is Tumstatin7-Peptide linker 7-CD137L1 (SEQ ID NO:35), 781 is Tumstatin7-Peptide linker 8-CD137L1 (SEQ ID NO:36), 114 is Tumstatin1-Peptide linker 1-CD137L4 (SEQ ID NO:43), 214 is Tumstatin2-Peptide linker 1-CD137L4 (SEQ ID NO:44), 314 is Tumstatin3-Peptide linker 1-CD137L4 (SEQ ID NO:45), 154 is Tumstatin1-Peptide linker 5-CD137L4 (SEQ ID NO:46), 254 is Tumstatin2-Peptide linker 5-CD137L4 (SEQ ID NO:47), 354 is Tumstatin3-Peptide linker 5-CD137L4 (SEQ ID NO:48). The results indicated that these representative recombinant proteins possessing activities of Tumstatin and CD137L could inhibit the proliferation of human umbilical vein endothelial cells, wherein SEQ ID NO:26, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:30 and SEQ ID NO:31 showed better inhibitory effects than positive control.

Example 5: Effects of the Bifunctional Recombinant Proteins Possessing the Activities of Tumstatin and CD137L on the Proliferation of Mouse T Cells

[0114] Mouse T cells were purified from single cell suspensions of mouse splenocytes using EasySep Negative Selection Mouse T Cell Enrichment kit (Stemcell), according to the manufacturer's suggested protocol. Purified T cells were plated at 10.sup.5 per well in 100 L medium and cultured in 96-well plates which had been coated with anti-CD3 monoclonal antibody at 7.5 g/mL overnight at 4 C. Four groups were designed: 1) Blank group without any stimulation (Medium+T cells); 2) the costimulation group in the presence of anti-CD3 (7.5 g/mL) and anti-CD28 monoclonal antibody (2.5 g/mL) (CD3/CD28); 3) the costimulation group in the presence of anti-CD137, anti-CD3 and anti-CD28 monoclonal antibody (CD3/CD28/anti-CD137mAb); 4) the costimulation group in the presence of recombinant proteins, anti-CD3 and anti-CD28 monoclonal antibody (CD3/CD28/Tumstatin1-CD137L). The cells were cultured in triplicate for 96 h. After initiating the cultures for 92 h, 10 L Alamar blue was added. Then, the absorbances of each well at 570 nm and a reference wavelength of 600 nm were determined by a microplate reader. The results are shown in FIG. 8, wherein 115 is Tumstatin1-Peptide linker 1-CD137L5 (SEQ ID NO:25), 155 is Tumstatin1-Peptide linker 5-CD137L5 (SEQ ID NO:27), 116 is Tumstatin1-Peptide linker 1-CD137L6 (SEQ ID NO:26), 215 is Tumstatin2-Peptide linker 1-CD137L5 (SEQ ID NO:37), 255 is Tumstatin2-Peptide linker 5-CD137L5 (SEQ ID NO:38), 256 is Tumstatin2-Peptide linker 5-CD137L6 (SEQ ID NO:41), 711 is Tumstatin7-Peptide linker 1-CD137L1 (SEQ ID NO:29), 721 is Tumstatin7-Peptide linker 2-CD137L1 (SEQ ID NO:30), 731 is Tumstatin7-Peptide linker 3-CD137L1 (SEQ ID NO:31), 741 is Tumstatin7-Peptide linker 4-CD137L1 (SEQ ID NO:32), 751 is Tumstatin7-Peptide linker 5-CD137L1 (SEQ ID NO:33), 761 is Tumstatin7-Peptide linker 6-CD137L1 (SEQ ID NO:34), 771 is Tumstatin7-Peptide linker 7-CD137L1 (SEQ ID NO:35), 781 is Tumstatin7-Peptide linker 8-CD137L1 (SEQ ID NO:36), 114 is Tumstatin1-Peptide linker 1-CD137L4 (SEQ ID NO:43), 214 is Tumstatin2-Peptide linker 1-CD137L4 (SEQ ID NO:44), 314 is Tumstatin3-Peptide linker 1-CD137L4 (SEQ ID NO:45), 154 is Tumstatin1-Peptide linker 5-CD137L4 (SEQ ID NO:46), 254 is Tumstatin2-Peptide linker 5-CD137L4 (SEQ ID NO:47), 354 is Tumstatin3-Peptide linker 5-CD137L4 (SEQ ID NO:48).

[0115] The results suggested that these representative recombinant proteins possessing the functions of Tumstatin and CD137L at a final concentration of 2 g/mL showed higher stimulatory effects on the proliferation of T cells than the costimulatory group CD3 plus CD28, which proved the significant synergistic effects between CD137L and CD28. Therefore, the representative recombinant proteins having the activities of Tumstatin and CD137L prepared in embodiments of the present invention possess good bioactivities in costimulating the proliferation of T cells.

[0116] It is understood that the examples and embodiments described herein are for further illustrative purposes only. Additionally, the examples showed only several types of ligations between different active fragments of Tumstatin and the extracelluar regions of CD137L. Actually, Tumstatin could but not limit to link the extracelluar regions of CD137L, and it could also couple with other proteins, peptides or just itself. It will be apparent that various other modifications and adaptations of embodiments of the invention will be apparent to the persons skilled in the art without departing from the spirit and scope of embodiments of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.