Synthesised the Vasonatrin peptide coding gene according to the Vasonatrin peptide amino sequence and Pichia pastoris codon preference, and then the 5' end of Vasonatrin coding gene containing His tag marker was obtained by polymerase chain reaction (PCR) technology. The spliced gene was inserted into Pichia pastoris secretory expression vector pPIC9K and pPICZαB, linearized separately. First of all, transformed the linear pPIC9K-VNP into Pichia pastoris GS115 by electroporation, screened the excellent transformants on G418 plates. Similarly, transformed the linear pPICZαB-VNP into GS115/ pPIC9K-VNP, and then selected the transformants on Zeocin plates. The expression efficiency of the GS115/pPIC9K-VNP/pPICZαB-VNP strains was analyzed by Tricine-SDS-PAGE and Western blot. The results showed that the yield is about 70mg/L in shake flask, which is increased by 75% compared with the single antibiotic marker screening method. And the research of biological activity indicated that the purified peptides can inhibit lipopolysaccharide (LPS) to induce iNOS and TNF-α in vitro macrophages.
Androgen receptor (AR) is a transcription factor belonging to Nuclear Hormone Receptor Superfamily and its DNA binding domain (DBD) is essential for its binding affinity and specificity to androgen response element. Traditional method to obtain the recombinant AR DBD is fusing it to glutathione S epoxide transferase or Staphylococcus aureus proteinA in order to improve the solubility and yield. The question is the fusion tag might affect the characterization of target protein, and removing of the fusion tag is usually cumbersome. In order to obtain functional AR DBD without fusion tag in an easy and convenient way, the sequence encoded human AR 520~644 amino acids which includes the DBD is amplified by PCR, and then inserted into pTWIN1 vector. After optimizing of the inducing temperature and IPTG concentration, almost all recombinant protein is expressed in a soluble manner at 20℃ and 30℃ in host E.coli strain BL21(DE3). The whole cell lysate is then loaded to Chitin beads and the optimal pH for self-cleavage is determined. Using this method the AR DBD without any fusion tags is purified by only one step and the product shows high purity and good performance in electrophoretic mobility shift assay.
Objective: To explore the effect of RPB5-Mediating Protein on cell genome stability and apoptosis of human hepatocellular carcinoma cell. Methods:RT-PCR was used to detect the expression of RMP mRNA on normal cell lines and several tumour cell lines. The SMMC-7721 cells and human hepatic cell HL-7702 cells were exposed to60Co γ-ray of different dose; then semiquantitative RT-PCR assay was used to detect the expression of RMP; Flow cytometric technique were used to detect apoptosis and cell cycle. To study the effect of RMP on cell genome stability and apoptosis of human hepatocellular carcinoma cell by RNA interfere . Results:RMP could express in different normal cell lines and tumour cell lines. The expression of RMP gene increased with the increase of exposure dosage. Flow cytometry results also indicated that cell apoptosis increased with increasing dosage, with the number of cells in G1 stage increased and that in S stage decreased. After RMP gene was interfered, the morphocytology of the cells changed obviously, meanwhile, the expression of p21 decreased. Conclusions:RMP may have a potential role on maintaining cell genome stability. RMP may associate with p53、p21, which have an role on regulating apoptosis.
In order to determine the basal expression and transcription activites of PDGFRβ promoterin two kinds of breast cancer cells MCF-7 andMDA-MB-231.The basal expression of PDGFRβ in MDA-MB-231 was identified by Western blot and Real-time PCR. Five deletion mutations of human PDGFR-β promoter region were obtained by PCR from human genomic DNA and then cloned into pGL3-basic vector based on Dual-Luciferase reporter system. Two human breast cancer cells MCF-7 and MDA-MB-231 were transfected by the recombinant reporters .Electrophoretic mobility shift assays(EMSA) was used to determine the DNA binding activities of transcription factors.As a result ,the region(+539bp,+1457bp) showed a positive regulatory role,while the(+54bp,+539bp)was negative.The(－983bp ,+54bp)was found to play a significant positive role in MDA-MB-231 whereas there was no effect in MCF-7.The transcription factor AP-1 was observed to highly express in MDA-MB-231 comparing with MCF-7.AP-1 was identified to bind the PDGFRβ promoter region and its binding was higher in MDA-MB-231 than in MCF-7.The fact demonstrate that basal expression and transcription activities of PDGFRβ promoter were different in two types of breast cancer cell and AP-1 played an important role in regulatory of PDGFRβ promoter in breast cancer cells.
NGR (Asn-Gly-Arg) motif exploited by phage display can selectively recognize neovasculature, and can be used for ligand-directed targeted delivery of various drugs and viral vectors to tumors or to other tissues with an angiogenesis process. Therefore,constructed the adenoviral vector Ad5/E1-mCherry/E3-luciferase-2A-eGFP/fiber-NGR which was modified with NGR peptide in the HI loop of the knob and could express three reporter genes. The study of the adenovirus in vitro and in vivo indicated that the three reporter genes could be expressed successfully and the infection efficiency indicated human breast cancer cell line MDA-MB-231 mediated by constructed adenovirus was higher than that mediated by unmodified control adenovirus Ad5CMVeGFP. The construction of the adenoviral vector laid a foundation for the further research on the target of adenoviral vector modified with NGR peptide and the therapy research of adenoviral vector carrying therapeutic genes in tumor animal model.
The objective is to construct a camelid nave single-domain heavy chain antibody phage display library. Total RNA was purified from 30ml blood of two healthy non-immune alpacas (Lama pacos) and directly used for complementary DNA (cDNA) synthesis. Three sets of primers were designed based on the conserved region of heavy-chain antibody. The repertoire of VHH coding sequence was amplified by nested PCR, and the PCR products were cloned into a phagemid vector pHEN1. By electroporation of E.coli TG1, the primary library (designate NAL) was obtained containing more than 107 independence clones. After helper phage rescue, the phage display library (designate SNA-PDL) was generated with a titre up to 1013 CFU/ml. The library exhibited high diversity as judged by the Hinf I restriction pattern. Solid phage biopanning against artificial antigen DON-MBSA showed significant enrichment of binding phage particles. The positive rate of panning round two was 36.4%. The data indicated that a nave single-domain antibody phage display library was constructed, which has good diversity and would be useful for generating VHHs with specific binding affinity.
The aim is to obtain strong promoters from Endophytic Klebsiella pneumoniae KKWB-5 of Banana, which will be applied to construct an endophytic engineered bacterium of banana. Promoter probe plasmid pUCK whose report gene is kanamycin-resistance (Kanr) gene was used to screen promoter fragments from KKWB-5 DNA in E.coli Top10. There were 7 Top10 transformants whose resistant level to kanamycin were over 2500μg/ml. This 7 plasmids were transformed into KKWB-5, and KKWB-5 transformants were tested their resistant level to kanamycin on LB plate and banana stem medium (BSM) respectively. After the plasmids were transformed into KKWB-5, the resistant level to kanamycin of KKWB-5 transformants when cultured on LB showed various degree of increases, but when cultured on BSM it weakened to a great degree. Transformant K-pUCK-15 was the highest resistant clone to kanamycin on BSM, and then the intergenic spacer region 15P of inserted fragment 15 were tested its promoter activity by pUCK as Kanr gene for report gene and by pUCK-6 as green fluorescent protein gene for report gene. The results demonstrated that fragment 15P had the same resistant level to kanamycin with fragment 15, moreover, the Top10 and KKWB-5 transformants containing recombinant plasmid pUCK-6-15Pgfp could emit intense green fluorescence under UV excitation when cultured on LB, and KKWB-5 transformants could emit green fluorescence under UV excitation too when cultured on BSM. In conclusion, fragment 15 had strong promoter activity in Top 10, but had stronger promoter activity in KKWB-5. The intergenic spacer region 15P was the main promoter region of Fragment 15, and it had good promoter activity in KKWB-5 when cultured on BSM. Fragment 15P can be applied to construct an endophytic engineered bacterium of banana.
CipA and CipB are two types of intracellular crystalline inclusion proteins produced by Photorhabdus luminescens bacteria, which are symbionts of entomopathogeic Heterorhabditis nematodes. To understand the biological function of these proteins for free living Panagrellus redivivus nematodes, recombinant Saccharomyces cerevisiae expression system of Cip proteins were constructed and the resulting yeast cells were used to feed the sterile J1 juveniles of P.redivivus. In recombinant yeast cells with CipA and/or CipB, the nematodes developed about 24 h faster than those in the yeast cells without Cip proteins. This promotion was reflected in two aspects: to short the cycle time and to enhance the reproductive ability of P. redivivus nematode. It means that the nutrient sources from entomopathogeic nematodes are acceptable by this free-living nematode.
The mature Y. lipolytica lipase Lip2 gene without the signal sequence and with a 6×His-tag in N-terminal,was ligated into vector pPIC9K. The recombinant plasmid pPIC9K-Lip2H was linearized by rectriction enzyme Sal I,and then transformed into Pichia pastoris GS115 by electroporation. A clone exhibiting a maximum lipase activity of 400U/ml in shaking flask was chosen for 10 L bioreactor cultivation. The fed-batch fermentation process was preliminarily optimized.With respect to the effects on biomass and the expression level of His6-YlLip2,the optimal basal salt medium was FM22,and the optimal parameters for induction pH and temperature were 5.0,25℃,respectively. After 114h methanol induction,the lipase activity of His6-YlLip2 reached the highest value of 3160U/ml. SDS-PAGE analysis showed that the molecular weight of the aimed protein was about 38 kDa. Using a Ni-NTA affinity chromatography,the purity of recombinant His6-YlLip2 reached 95.43% by one-step purification with the specific activity of 4250U/mg.
Selective medium with sodium carboxymethyl cellulose (CMC) as only carbon source was employed for screening T-DNA insertional library of Aspergillus niger, from which mutant AN-108 with low cellulase activity was isolated. Its enzyme activity is 83.3% to that of wild type Aspergillus niger strain. Solid state fermentation analysis showed that no cellulase activity difference was obtained between mutant AN-108 and wild type strain of A. niger. It’s hypothesized that sugars existed in solid state fermentation media might contribute to the restoration of cellulase activity of the mutant. To test this possibility, CMC media with different sugars were used for culturing mutant AN-108. Hydrolyze zones around colonies were observed in all plates with sugars. The experiment result suggested that sugar might function as expression inducers overcoming the mutation effect. To identify the gene disrupted in mutant AN-108, sequence analysis of T-DNA insertion site was performed using inverse PCR method. The result of sequence blast indicated that the disrupted gene had 90% identities to the gene An14g03730 of A. niger, which encoded a proline-rich protein.
One clone (LIP001) producing cold-active lipase was selected from metagenomic library of deep sea sediment for the optimization study of fermentation conditions. The effects of main factors on the growth and lipase-producing of LIP001 were determined by single-factor test. The results showed that the optimal condition were 30℃, pH7.0, and inoculum size 5%, liquid volume 50ml in 250ml flask, respectively. The fermentation conditions for the lipase-producing of LIP001 were further optimized by orthogonal experiment on the basis of single factor test. The major factors and their concentrations were as follows: olive oil (1%), yeast extract(0.5%), peptone (1%), (NH4)2SO4 (0.5%), phosphate (0.5%), MgSO4 (0.2%), and chloromycetin(12.5μg/ml), respectively. Under the optimized condition, the lipase activity was 1980U/ml which was increased by 54.7%. For 5L fermentor research, the lipase activity was 2420U/ml.
Objective: To expresss and purify the aflatoxin-detoxifizyme(ADTZ) in E.coli. and to study the biological activities and secondary structure of rADTZ. Methods and Results: the mature peptide of ADTZ was subcloned into pMAL-c2x vector to construct the prokaryotic expression plasmid. The recombinant plasmid was transformed into Rosetta(DE3) and the soluble fusion protein MBP-ADTZ was highly induced by IPTG. The expression level was approximately 50% of the total bacterial protein. The pure fusion protein was got from the cell lysate by amylose affinity chromatography. 42kDa MBP and 76kDa rADTZ were gained via digestion of fusion protein by Factor Xa, and pure rADTZ was obtained by Hydrophobic interaction chromatography(HIC). Biological activities of rADTZ had been examined and results showed that the protein had AFB1-detoxifying activity with the specific activity of 136U/mg. Circular dichroism spectra analysis revealed that rADTZ was composed of 43.3% of α-helix, 31.1% of β-sheet, 10.5% of β-turn and 15.1% of random coil. Conclusion: Purified rADTZ protein with AFB1-detoxifying activity was obtained and that laid the foundation for exploration of the relationship between the structure and function of rADTZ.
Gentamicin producing strain Micromonospora purpurea was treated with 15μg/ml penicillin when M. purpurea had cultured 36h, and the cells that had cultured for 48h at logarithmic growth phase were treated with UV and NTG, then they were cultured in incubator at 37℃ for 5~8 days, and when the colonies grew out, the cells were treated by UV again. As a result, a high yield strain N-14 was obtained. Compared to control, the biological titer of N-14 fermented in tube was increased from 625u/ml to 1913u/ml, and the biological titer of N-14 fermented in flask was 2178u/ml and increased by 75.2% compared to control 1243(u/ml). Besides, the reducing sugar, total sugar, amino nitrogen, pH, cell concentration and other metabolic parameters were determined during fermentation process in 5L fermentor, and the culture condition was researched. The average biological titer of 5 batches was 1967u/ml and was increased by 68% compared to control 1171u/ml.
Urate oxidase (uox, C 1.7.3.3) was responsible for the oxidation of uric acid to allantoin.The urate oxidase gene from Aspergillus flavus was cloned into expression vector of pET43.1a. The sequence was confirmed by DNA sequencing and double endonuclease digestion, and transformed to competent cell named JM109. Recombinant strain was induced to express urate oxidase protein,and the target protein was almost the soluble protein after sonication;the uox pure was purified from supernatant after anion and cation column two-step column purification;the activity of pure uox was determined by spectrophotometry. The results showed that: urate oxidase was high level expressed in E. coli, and the ratio of uox to bacterial proteins could reach 50%; expression product was purified by two steps of chromatography and purification method is simple to obtain high purity protein; the protein had the activity of breaking down uric acid in vitro.
In order to achieve efficient fatty acid accumulation in microorganisms,a continuous supply of acetyl-CoA directly in the cytosol of the cell should be satisfied. Considering that acetyl-CoA is the precursor in both the fatty acids synthetic and mevalonate pathway, the mevalonate pathway was inhibited and then more acetyl-CoA might be channeled to the fatty acids synthesis. A method of addition of precursor or/and the inhibitors of mevalonate pathway were used to reinforce acety-CoA supply. Acetate acid is the putative precursor of acetyl-CoA. Simvastatin and citrate acid are the inhibitors of mevalonate pathway. The effects of acetate acid, simvastatin and citrate acid on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. were investigated. Including addition of acetate acid at the beginning or/and in the late period of fermentation, addition of simvastatin or citrate acid, the supplementation with acetate acid and simvastatin or citrate acid at the beginning of fermentation. The results showed that the highest of DHA yield reached 13.21g/L by combinations of 6mmol/L acetate acid and 1μmol/L citrate at the beginning of fermentation, which was 46.61% higher than that of control.
The effects of different concentrations of carbon and nitrogen sources on recombinant E. coli BL21(DE3) on the fermentation of sucrose isomerase were investigated,the culture media of recombinant E.coli producing sucrose isomerase was optimized using Design Expert 7.1.6 software combined with the Plackett-Burman design and central composite design method.The results showed that the optimal media for culture as follows:Cane molasses 10.65 g/L ,Corn steep 22.22 g/L, NaCl 7.57g/L ,MgSO4·7H2O 0.52g/L, KH2PO4 4.46g/L.It is found that the Sucrose isomerase activity was 29.1U/ml, compared to LB medium culture E.coli(15U/ml), sucrose isomerase activity increased by 94%, with the original strain increased 21.4 times(1.3U/ml) compared.
Twenty strains of freshwater and marine microalgae collected in our laboratory which belong to Chlorophytes, Eustigmatophytes and Bacillariophytes were screened for their potential of oil production. All microalgae were cultured in batch, with bubble column photobioreactor (light path, 3 cm). These microalgae were evaluated by measuring dry weight, total lipid content, total lipid yield, total lipid volumetric productivity. The results showed that the dry weight and total lipid contents of twenty microalgae were in the range of 1.81~7.88g/L and 16.0%~55.9% dw, respectively, and nine of them were referred as oleaginous microalgae, including Scenedesmus sp.(6.34g/L, 55.9% dw), Chlorococcum tatrense(5.93g/L, 46.9% dw), Nannochloropsis oculata(7.88g/L, 35.0% dw), Chlorococcum oleofaciens(5.58g/L, 45.9% dw), Pseudochlorococcum polymorphum (6.10g/L, 40.0% dw), Chlamydomonas augustae(5.78g/L, 40.5% dw), Chlorella ellipsoidea(5.56g/L, 40.7% dw), Chlorococcum ellipsoideum(5.41g/L, 38.0% dw), Chlorococcum nivale(5.55g/L, 36.3% dw). It was concluded that the most potential for industrial application was Scenedesmus sp, which lipid yield and lipid volumetric productivity was 3.5g/L and 218.7mg/L·d, respectively.
A modified and simple manufacturing scale process for the purification of plasmid DNA was developed with low cost. The Escherichia coli JM108 host strain was selected for the fermentation with protein free media because of its advantage to produce predominantly super coiled plasmid DNA. The alkaline lyses method was used. The plasmid purification process including chromatography-absorbent silica, suitable DNA binding solution, endotoxin-removal reagents was substantially modified. The tests for the quality control were established for the detection of the residue impurities. The purity of the plasmid DNA obtained by this process can meet the clinical quality requirements.
The establishment of an efficient adventitious shoot regeneration system from leaf discs and stem segments of one-year-old Russian olive (Elaeagnus angustifolia L.)was established. Shoot regeneration using leaf explants and stem segments was obtained on MS medium supplemented with different cytokinins viz., 6-benzyladenine (BA) and Zeatin (Zt), with combination of auxins viz., naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA). MS + 0.5mg/L 6-BA + 0.2mg/L NAA was found to be a better combination for leaf regeneration, producing a maximum of 4.3 shoots per explant, whereas MS + 1.0mg/L Zt+0.5mg/L NAA for stemsegment explants produced the highest number of multiple shoots (3.6) per explant. The regenerated shoots produced 100% rooting on 1/2 strength MS medium supplied with 0.5mg/L NAA. In vitro raised plantlets were successfully transferred to pots containing sterile soil∶peat moss∶sand (1∶1∶1) for acclimatization， and 77% of rooted plantlets survived under ex situ conditions. The currently employed regeneration protocol through leaf explants and stem segments facilitates germplasm conservation of this medicinally and ecologically important species besides providing a standardized regeneration protocol for genetic transformation studies in the future.
Plasmid DNA encoding treatment gene for pharmaceutical applications was approved into the clinical trials. A pre-requisite to the success of plasmid-based therapies is the development of effective production of plasmid DNA. However, at present, there are several problems and bottlenecks in large-scale production of pharmaceutical plasmid DNA. Such as vector construction, cell lysis, bacteria chromosome DNA removing, bacteria endotoxin removing and quality-control in production process. The downstream for the large-scale production of plasmid DNA, limitations and the strategies used to obtain a final product that meets specifications was reviewed.
Porcine reproductive and respiratory syndrome,characterized by reproductive failure and respiratory disease in pigs, is a major disease in the swine industry worldwide. Currently,its pathogenic mechanisms and self-replication pattern are not entirely clear, so it hasn't been removed from the fundamental existence of the disease,then the detection of PRRSV is particularly significant.Various detection methods had been introduced,for example, the intuitional clinical observation,precise laboratory differential diagnosis and so on.Among them,the laboratory molecular biological diagnosis technique had been described in detail,and it is summarized by laboratory diagnosis and common biological diagnosis.
Recently, some researches suggested that cereals contain proteinaceous inhibitors of endo-xylanases, decreasing the practical effect of xylanase in animal feed industry. Since this kind of inhibitor was found firstly from wheat, a large number of endo-xylanase inhibitors have been discovered. They are mainly divided into three classes: xylanase inhibitor protein (XIP) type,Triticum aestivum xylanase inhibitor (TAXI) type and thaumatin-like xylanase inhibitor (TLXI) type. These three different types of xylanase inhibitors profiles were reviewed, and a theoretical basis for further study on inhibition mechanism of xylanase inhibitor and molecular improvement on xylanase were provided.
