APPL proteins, including APPL1 and APPL2, are important messengers between cell membrane and cell nucleus and are essential to cell proliferation. In this study, we expressed and purified the PTB domain of human APPL2 (hAPPL2) in Escherichia coli. Phage display technology was then used to screen for the interacting peptides with this domain. After 3 rounds of panning, 48 single phage plaques were randomly picked and ELISA analysis was performed. Two 7-mer peptides, ERLPFFY and YLTSPKH were found to bind to the PTB domain of hAPPL2 in a specific manner. Both wild type and mutant forms of P3-GST fusion protein, with each amino acid substituted by alanine, were prepared and evaluated for binding capacity with hAPPL2 PTB by ELISA. Results showed that each residue was indispensible in the binding between this peptide to hAPPL2 PTB domain. This study shed light on structural interactions between the PTB domain of APPL2 and the binding proteins.
AbstractObjective:To evaluate the ability of the porous nanohydroapatite crystals and polyamide composite (nHA/PA66) as scaffold compound with rhBMP2 to repairing bone defect, study the methods which fast the artificial bone with host bone healing in vivo.Methods: The animal models of bilaterial radius bone defect created with surgery in the New Zealand white rabbits, and were implanted with nHA/PA66/ rhBMP2 as experimental group, implanted nHA/PA66 as experimental control group, and no implant as blank control group. The effect were observed by gross Xray and histopathlogical examination and in situ hybridization at1, 2, 4, 8, 12W after operation, and use the spss12.0 and OneWayANOVA system to analysis. Results: The defects of experimental group and experimental control group were repair perfectly, The blank control group hadn't repaired after 12weeks, the speed of bonehealing of nHA/PA66/ rhBMP2 group were faster than nHA/PA66 group in the early time. The data of in situ hybridization analysis had statistically significant difference between nHA/PA66/ rhBMP2 and nHA/PA66 group in two weeks, but the data had no statistically significant difference between nHA/PA66/ rhBMP2 and nHA/PA66 group in four weeks . Conclusion:The results show porous scaffold of nHA/PA66 compound with rhBMP2 can enhance the ability of the artificial bone with host bone healing in the early time in vivo.
DEK protein’s carboxyterminal DNAbinding region (CBD) is a newly found DNAbinding domain of DEK,which contains several phosphorylation sites and has a close correlation with DEK protein's function in vivo and in vitro.Using prokaryotic expression system,the peptide of DEK protein's carboxyterminal DNAbinding region (CDB) was expressed and purified.In detail,the CDB DNA fragment was constructed into pET30a (+) vector,and E.coli BL21 (DE3) competent cells were used as host cells.The fusion protein HisCBD was expressed by induction of IPTG and purified by NiNTA agarose.The result of SDSPAGE showed that the molecular weight of the purified protein was about 10.7kDa.Electrophoretic mobility shift assay (EMSA) indicated that DEKCDB prefered to bind to supercoiled form of DNA in vitro,it had similar character to the binding of whole length DEK protein with DNA.This suggested that the carboxyterminal DNAbinding region of DEK protein might function on the binding of DEK protein to DNA partly.
To reduce the clearance of sGLP-1, sGLP-1-HSA fusion protein was constructed by the fusion of the C-terminus of sGLP-1 to the N-terminus of HSA via an 6 amino acids linker and expressed in the pichia pastoris.Screen the strain with resistsnce of 4g/L G418. After fermentation in a 5L bioreactor，the expression of sGLP-1- HSA was induced by methanol.After 48h, the expression level reached 0.8g/L.The sGLP-1- HSA was abtained following a series of purification steps such as ultrafiltration, sephadex G-25 and blue- sepharose. The purity of the fermentation product could reach 95% by means of HPLC，and the total recovery yield could reach 60%.The biological activity of sGLP-1- HSA in GKⅡ diabetes rat and healthy rat suggested that the plasma glucose level was significantly lower after subcutaneous administration of sGLP-1- HSA..Expression of sGLP-1- HSA fusion protein can provide foudation for abtaining a larger quantity of recombinant sGLP-1- HSA for experimented and clinic studies.
In the present work we have studied if Eudragit S-100, a pH-responsive polymer can enhance the refolding level of TGF-β1 (Transforwing growth factor，TGF-β1). The refolding of TGF-β1 was performed by directly diluting denatured TGF-β1 into a refolding buffer containing different concentration of Eudragit. The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTT assay, reverse phase HPLC, fluorescence emission spectroscopy and circular dichroism spectroscopy. The addition of Eudragit S-100 in the refolding buffer significantly increases TGF-β1 refolding yield to 53%, when dilution refolding was conducted at 0.5 mg/ml TGF-β1. This study shows evidence of an electrostatic interaction between oppositely charged TGF-β1 and the Eudragit polymer during refolding. This ionic complexing of Eudragit and TGF-β1 appears to shield exposed hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic-driven aggregation of the molecules.
To clone human truncated TGF-β receptor II（tTGF-βRⅡ）and express it in Escherichia coli. The gene tTGF-βRⅡ,obtained by PCR was cloned into the vector pGEX4T3 to construct a fusion expression plasmid.The recombinant protein GST-tTGF-βRⅡ were expressed in soluble form, purified by Glutathione-Sepharose 4B affinity chromatography and cleaved by thrombin to release tTGF-βRⅡ. Purification of tTGF-βRⅡ was achieved by affinity chromatography. SDS-PAGE indicated that fusion protein GST- tTGF-βRⅡ was secreted as a protein of around 37.0 KDa and aimed protein tTGF-βRⅡwas 11.0 KDa. The GST-tTGF-βRⅡand tTGF-βRⅡ protein were confirmed by Western blot analysis. The purification of aimed protein was biologically active in a validated inhibited human fibroblast proliferation bioassay.
The sequence of mink IGF-Ⅰgene was obtained and analyzed,and the fusion protein of the IGF-Ⅰ was produced in E.coli. By RTPCR,liver IGF-Ⅰfrom mink were isolated. The IGF-ⅠcDNAs encoding 153 amino acids,and showed high degree of homology(>90%)with other reported sequences(gold monkey,lesser panda,giant panda,A.tiger,horse,et al.) at nucleotide and amino acids levels.Two differences in mink amino acids sequences were found from sequence alignment.Whether these differences affect the molecular conformation or function for the growth factor's processing or these changes in the molecular structure may be responsible for the abnormal ability of growth and reproductive by influencing the function of IGF-Ⅰ have not yet been determined.The DNA fragment encoding the mature peptide of mink IGF-Ⅰwere subcloned to the pGEX-6P-1 expression vector and highly expressed in E.coli.BL21 with IPTG induction. The expression fusion proteins(pGEX-6P-1 IGF-Ⅰ)were mostly existed in soluble form and were about 34kDa.The induced cells culture were proved to be active IGF-Ⅰantigens. The mink IGF-Ⅰ gene was successfully cloned and expressed.Furthermore,the structure and function of this gene were analyzed and predicted. These result provided a good basis for further study of its biological activity.
Fgf9 is the most important signal factor implicated in mediating the proliferation of Sertoli cells and the cord formation in testis morphogenesis of vertebrate. With the cloning principle of expressed sequence tags, sequence splicing and RTPCR were applied in the cloning of novel gene from Holstein cow and analyzing its expression pattern in various cow tissues. The bioinformatics analysis on the sequence and protein results of Fgf9 was conducted. Results indicated that Fgf9 was located at cow 12 chromosome, with fullsequence of 697bp. The open reading frame is 627bp in length and encodes a deduced amino acid sequence of 208 residues. The molecular weight of Fgf9 protein is 23.38245kDa, and its pI is 7.0600. RTPCR demonstrated the correctness of its ORF. Fgf9 was expressed in each tissue. No obvious homology with other cow cDNA was found for Fgf9.The GenBank accession number EU693028 was achieved. Function and structural analysis indicated that Fgf9 has a typical FGFs conserved domains, receptor interaction site and heparin binding site. It was predicted that Fgf9 has no signal peptide.
To elucidate structural characters and expression characters of RGDV P8 protein in E.coli, bioinformatics analysis were performed by using biology software and analysis tools online.P8 gene was successfully amplified by RT-PCR and inserted to pGEX-4T-1 expression vector. The protein was then expressed in E.coli BL21(DE3) with IPTG induction. The antiserum was produced by immunizing rabbit with fusion protein, and the specificity of antiserum was evaluated by detecting RGDV, RDV, RSV, RRSV. Analysis showed that P8 protein had a typical transmembrane helices region, and a Phytoreo P8 domain shared with Phytoreo P8 family. SDSPAGE and Western blotting analysis showed that molecular weight of P8 fusion protein was about 73 kDa. P8 protein was efficiently expressed in the form of inclusion body. The antiserum with high titer and specificity provides the basis for detecting RGDV and studying function of P8 protein.
The two pabAB genes encoding aminodeoxychorismate synthase(ADC synthase) from a Lserine producing strain Corynebacterium glutamicum SYPS062 and model strain Corynebacterium glutamicum ATCC 13032 were ampilified by PCR. The result of nucleotide sequence analysis showed that both pabAB fragments were 1863bp, encoding 620 amino acids. 16 bases differences that resulted in the changes of 7 amino acids were found in the pabAB of SYPS062.The two pabAB were inserted into pET28a to yield the recombinant expression vector pET28apabAB and then transfromed into BL21(DE3).Upon IPTG induction,soluble ADC synthase was overproduced by E.coli BL21(DE3) harboring the expression construct.Recombinant ADC synthase purified by NiNTA affinity chromatography showed a single band about 67kDa on SDSPAGE gel, and activity of aminodeoxychorismate synthase analysis show that the enzyme specific activity of SYPS062 is 46.6% lower than ATCC 13032.
The protoplasts of Micromonospora purpurea, the high yield strains of gentammicin producer were mutagenized by diethyl sulfate (DES) and ultraviolet radiation (UV)respectively， then fused, screened by gentamicin resistance and regenerated. The average fermentation unit 2200±U/ml could be achieved by shake flask for 10 batches. The average fermentation unit 1900±U/ml could be obtained by 5L fermentor for 7 batches. The quality of the end product conformed to CP2000, BP2000 and USP26 pharmacopoeia.
Gene deletion vector pLJ04(pKC1139∷△bkdF+△bkdH)was used to disrupt bkdFGH in Streptomyces avermitilis 76-02-e, an industrial producer of anthelmintic avermectin. The disruptants were confirmed by PCR. Shake flask experiment and HPLC analysis showed that the mutant lost the ability to produce avermectins. As it is expected, the mutant, named S.avermitilis bkd76-3, could restore the ability of producing avermectins when the feeding of methylbutyric acid and isobutyric acid to its fermentations was carried out. The addition of cyclohexanecarboxylic acid (CHC) into fermentations of the S.avermitilis bkd76-3 allowed for production of four components, two of which was confirmed as CHC-B1 and CHC-A2 by LC/MS analysis, respectively.
Objective：Addition of gold nanoparticles (average diameter10nm) into amplification system with low copy gene fragments from the complicated genome helps to enhance the specificity of PCR amplification. Methods：PCR amplification model system of the complicated genome was simulated, PCR was performed with approximate single copy λ DNA as the template and addition of gold nanoparticles. The experiments were so repeated and optimized that the PCR amplification model system was built by simulating the PCR amplification of the complicated genome with low copy gene. Then, the low copy gene (tumor necrosis factor, TNFα gene exon 1 with 380bp) of human genome was amplified so as to verify the actual efficiency of enhancement of the specificity of PCR amplification by the gold nanoparticles. Results：The gold nanoparticles could better enhance the specificity of PCR amplification in the PCR amplification of the complicated genome system with low copy gene. Conclusion：It was preliminarily indicated that nanoparticle PCR method based on gold nanoparticles could optimize the PCR amplification of the complicated actual genome system with low copy gene, which made an important valuable reference to improvement and extension of the optimized methods of PCR amplification.
Pseudomonas putida isolated relieving salt stress and promoting plant growth was used as the recipient strain. Optimum conditions including growth stage of the strain, concentration of competent cell, electroshock voltage and the meduim were investigated for the electroporation of P.putidaE.coli shulter vector PDSK519 into P.putida Rs198. It was showed that the highest transformation efficiency was up to 1.3×107CFU/μ g DNA in the optimium condition, under which the competent cells were collected at logarithmic growth phase(OD600=0.5), the mixture of the competent cells(4.6×1012/ml) and PDSK519 plasmid DNA was electroporated at 13kV/cm. It will be very helpful for developing a genetic transformation system and studying the genomic function of P.putida.
Sugarcane stem is an ideal organ for producing foreign pharmaceutical proteins and chemicals by genetic engineering. A perfect promoter driving foreign gene to express strongly and specifically in sugarcane stem is necessary for this purpose. In order to isolate a Sugarcane stemspecific promoter , a fragment of 1968bp nucleotide sequence（Ppst2a）upstream 5′ of sugarcane pst2a gene, which was demonstrated to express specifically in sugarcane stem previously was isolated by using chromosomal walking. Bioinformatical analysis of this sequence shows that the sequence contains some typical elements of a promoter. To identify the stemspecific of this promoter, a construct was derived from pCAMBIA1301, which original CaMV 35S promoter was replaced by the 1968bp nucleotide sequence, and named as pCAMBIA1900. Transformations of pCAMBIA1900 and pCAMBIA1301 to leaves and stem pieces of sugarcane were carried out by using particle bombardment. The transient expression of gus showed that the gus expressed specifically in sugarcane with a little higher level compared with CaMV 35S. It is the first report that pst2a promoter is a potential stemspecific promoter which can further be used in transgenes into sugarcane.
The high production inulase strain was screened from the soil sample where burdock planted in Qin Village, Bayou Town, Pei County, Xuzhou. Inulase activity were determined which produced by 40 strains separated from soil. Three mold stains, C122803、D081506 and D081513, which had higher ability of producing inulase were obtained by using transparent circle method as initial screening and rocker method as rescreening. Enzyme activity of the three strains were 1.411U/ml, 1.895U/ml, 1.792U/ml,separately . Enzyme activity of D081506, 1.895U/ml, was the highest. The fermentation conditions of D081506 were studied and the optimized conditions were lappa juice 2.0%, yeast extraction 1.6%, (NH4)2SO4 0.5%， NaCl 0.5%，K2HPO4 0.5% and pH 5.0. Inulase activity of D081506 was 2.9578U/ml which increased 56.09% under the condition of 27℃,140r/min, 24h.
Recombinant antibody drugs has developed through mice monoclonal antibody（McAb）, humanmice chimeric antibody, humanized antibody and full human antibody. They have been applied in clinical immunotherapy for the treatment of cancer, autoimmune diseases, viral diseases and Alzheimer disease, etc. The two basic principles in antibody humanization are keeping or enhancing affinity and reducing immunogenicity of engineered antibodies. The degree of humanization has approached 100% following the continuously improved techniques, such as CDR grafting, resurfacing, antibody library and transgenic mice. However, the gap between the basic research and clinical application still need to be filled. The outline, challenge and perspective in application associated to recombinant antibody drugs were reviewed here.
The tandem expression vector can effectively increase the expression level and structure stability of the target peptides (protein) with small size of molecular, and avoid toxicity towards host cell from some toxic peptides product, therefore, this approach is widely applied in biotechnology. The four construction methods of tandem expression plasmid including asymmetric and complementary cohesive ends, directional adapter, isocaudarners, and tandem expression cassette were reviewed in terms of protocol, characteristic and applicable field. In addition, selection principles from various construction methods of tandem plasmid were reviewed in terms of efficiency, accuracy and product cleavage.
Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which a pair of proximity probes are designed to bind pairwise to target proteins via a DNA ligation reaction of oligonucleotides results in the formation of an amplifiable DNA strand suitable for analysis. It allows extremely sensitive and specific detection of proteins by allowing the protein targets to be represented as DNA signatures that may be subsequently amplified and detected by methods like PCR. Here the basic principle and the potential applications of PLA were discussed, as well as the preparation of proximity probes.
DNA molecular weight standards are a mixture of a grou Pof DNA molecules with known sizes to reference the molecular weight (mass) of unknown DNA samples in nucleic acid electrophoresis and hel Pthe experimentalists to judge the property of the DNA samples. At the present time the DNA molecular weight standards are essential for nucleic acid electrophoresis analysis in the molecular biology and genetic engineering fields. The methods and principles for the production of DNA molecular weight standards with the recent advances in this field were summarized.
Induced pluripotent stem cells (iPS) are derived from the differentiated somatic cells that regain the differentiation capabilities by the direct nuclear reprogramming with the exogenous factors. The methods for iPS induction evolved from transcription factors to RNA binding proteins, small molecules, and extracellular signals with the efforts in improving biosafty. The cellular and epigenetic characterization for iPS generation is a progressive and timedependent process, which is tightly associated with the cellular differentiation status. However, epigenetics of iPS is not same as the embryonic stem cells. In combination with gene therapy and cellular transplantation therapy, the achievements of iPS had been applied in animal disease model treatment.
With the development of industrialization, the problem of oilcontamination to the soil is getting more and more serious. How to clear up or remove the oilcontaminants from the soil becomes an important environmental problem for all countries around the world. Bioremediation, as the methods with fast and safe in processing, low cost and nonsecondary contamination, is becoming the main solution to soil environment by oil contamination. Biostimulation and bioaugmentation are most commonly used techniques in bioremediation.The theory of bioremediation, including the concept and method of biostimulation and bioaugmentation were introduced, and advance study and progress in this field from the world in recently years were demenstrated. Both of the two methods can lead a significant decrease in soli TPH content, but the efficiency relates to many factors. Accordingly, the bioremediation technique should be tailored specifically to each polluted site.
