Fusion Expression of Glucoamylase and Xylanase in Pichia pastoris

Employing RT-PCR amplification method, the mature pepfide coding sequence of glucoamylase （amyA） gene was amplified from total RNA of Talaro- myces emersonii and the xylanase （xyrut） gene from genomic DNA ofPaenibacillus sp. H10-3. The result showed that the mature peptide sequence of amyA is 1857 bp long and encodes 618 amino acids; and the mature peptide sequence of xynA is 636 bp long and encodes 211 amino acids. Then these two genes were spliced by overlap extension PCR （ SOE-PCR）, yielding fusion gene amyA-l-xynA. The fusion gene amyA-l-xynA was then cloned into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid pPIC9-amyA-l-xynA. The recombinant plasmid pPICg-amyA-l-xynA was linearized and transformed into Pichia pastoris GSll5 via electric shock, yielding engineering strain ALX2. The maximum yields of glucoamylase and xylanase in ALX2 fermentation supernatant were determined as 10.7 and 51.8 U/ml, respectively.

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1.Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR),then cloned into vector pET32a(+),and expressed as thioredoxin-PedA fusion protein in the host strain E.coli BL21 (DE3).The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column.Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase.Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E.coli (pPA003PED1) using Listeria monocytogenes as the indicator strain.Thioredoxin-PedA fusion gene was further cloned into pET20b(+).Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces.The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E.coli (pPA003PED2).Therefore,biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.

Expression of Mouse MT-1 as a Fusion Protein in Anabaena sp. PCC 7120

To produce mouse metallothionein-1 (mMT-I ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia co/i-cyanobacterium shuttle fusion expression vector, pKG-MT, was constructed. Via this vector, mMT-I cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione-S-trans-ferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS-polyacrylamid gel electrophoresis (SDS-PAGE) showed that the fusion protein GST-MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio-β-D-galactoside (IPTG). Glutatione-S-transferase metallothionein (GST-MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT- I was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G-50. SDS-PAGE demonstrated that the purified mMT- I was the desired protein. The result of ELISA for the purified mMT-I showed that the recovery of mMT- I from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal-binding activity of the purified mMT-I was almost the same as that of wild type MT.

Expression, purification and characterization of a phyA^m-phyCs fusion phytase

The phyA^m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile ofphytase and decrease the cost of production. The fusion phytase phyA^m-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is （25.4±0.53） U/ml at the flask scale and （159.1±2.92） U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 ℃ and an optimal pH at 5.5-6.0 and its relative activity remains at a relatively high level of above 70% in the range ofpH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 ℃ to 95 ℃ for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE （sodium dodecyl sulfate-polyacrylamide gel electrophoresis）. After deglycosylation by endoglycosidase H （EndoHf）, the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those ofphyCs andphyA^m.

MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF α-BUNGAROTOXIN (V31) FROM CHINESE CONTINENTAL BANDED KRAIT

The cDNA encoding a variant of α bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino acid mature protein.The signal peptide is very similar to those of short chain neurotoxins,κ neurotoxins and cardiotoxins.The amino acid sequence of the mature protein is identical to α bungarotoxin (V31),a minor variant of α bungarotoxin identified by protein sequencing technique.Furthermore,the cDNA encoding the deletion precursor of α bungarotoxin was also cloned.By use of pMAL p2,the variant was overexpressed in E coli as a soluble fusion protein and purified by sepharose 6B amylose affinity chromatography,which was confirmed by western blotting with the antisera agai nst α bungarotoxin.The recombinant variant was achieved after digestion by factor X a.It displayed about 1/6 in vivo toxicity of natural α bungarotoxin.The successful cloning and functional expression of α bungarotoxin provided a basis for the future study of structure function of long neurotoxins.

Gene Cloning and Expression of PKA Catalytic Subunit and Its Activity in Arabidopsis thaliana

Protein phosphorylation and dephosphorylation are the general means of regulation metabolism within a cell. A PKA catalytic subunit was found in Arabidopsis genome using Blast software. The cDNA was cloned by RT-PCR and sequencing result indicated a high degree of homology at protein level. The cDNA was subcloned into pET30a (+) and expressed in E. coli at different temperatures. The target protein was insoluble when induced at 37degreesC, while dissolvable if induced at 22degreesC with 0.01 mmol/L IPTG. Ni2+-NTA affinity chromatography was used to purify the target protein, which was shown to have cAMP-dependent protein kinase activity. Western blotting analysis indicated that stress treatments affected the expression of PKA catalytic subunit at protein level just to a small extent.

Prokaryotic Expression and Potential Application of the Truncated PCV-2 Capsid Protein

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 （PCV-2）. The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a（＋） vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta（DE3） E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % （73/264）. Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-20RF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.

Prokaryotic Expression of Antimicrobial Peptide CATH PR1–2 from the Skin of Paa robertingeri in Escherichia coli

The aim of this study was to investigate the prokaryotic expression of antimicrobial peptide cathelicidin （CATH） PR1 and PR2 from the skin of Paa robertingeri in Escherichia coli. Two active peptides, CATH PR1 and CATH PR2, belong to the CATH family in the skin of P. robertingeri. CATH PR1 has a relatively high antimicrobial activity, especially for the drug-resistant strains found in clinical practice; however, no antimicrobial activity has been found in CATH PR2. The molecular weights of both CATH PR1 and CATH PR2 are relatively low （3195.88 and 2838.34 Da, respectively）. Thus, the genetic processes, as well as the expression and purification of these proteins, are difficult to perform. Therefore, in this study, CATH PR1 and CATH PR2 genes were tandem ligated and then connected to the plasmid pET-32a. This reconstructed plasmid was then transfected into the expression vector E. coli BL21 to construct the recombinant expression system. The fusion expression of peptide PR was stable in E. coli after induction with 1.0 mol/L isopropyl β-D-1-thiogalactopyranoside at 37℃ for 4 h. The antimicrobial activity assay using Staphylococcus aureus （Song） and Candida albicans 08030102 showed that the antimicrobial activity of PR was similar to the antimicrobial activity of CATH PR1. This study showed that artificial modification of the amino acid sequences of PR1 and PR2 could result in better protein expression in prokaryotes, and the fusion protein expressed had relatively high antimicrobial and other biological activities. In conclusion, the findings suggest future prospects of the commercialization of this method.

Advances in Antimicrobial Peptides and Expression Strategy

Antimicrobial peptides are widely distributed in nature,existing in organisms of plants,insects,and vertebrates.It has been approved that antimicrobial peptides have broad spectrum antimicrobial activities,and play a key modulatory role in the innate immune response and tumor inhibiting activity.Due to the special action mechanism,the antimicrobial peptides become a hot field of genetic engineering.In the present paper,the general properties,mechanism of action,application value,existing problems,the latest progress and the expression strategy were discussed.

A Sensitive and Specific IgM-ELISA for the Serological Diagnosis of Human Leptospirosis Using a rLipL32/1-LipL21-OmpL1/2 Fusion Protein

Objective To construct a lipL32//1-1ipL21-OmpL1//2 fusion gene and its prokaryotic expression system, and to establish an enzyme-linked immunosorbent assay （ELISA） using the rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for sensitive and specific detection of IgM in the serum of patients with leptospirosis. Methods lipL32/1-1ipL21-OmpL1/2 fusion genes were constructed using a primer-linking PCFI. The target recombinant protein antigens, rLipL32/1, rLipL21, rOmpL1/2 and rLipL32/1-LipL21-OmpL1/2, were expressed and the purified antigens were then immobilized to the surface of microplate wells for ELISA-based detection of IgM in the sera of leptospirosis patients; Results Of 493 acute leptospirosis patients, 95.7% and 97.8% were positive by rLipL32/1-LipL21- OmpL1/2-1gM-ELISA using different serum dilutions, which was higher than the rLipL32/1-1gM-ELISA （93.1% and 90.3%）, rLipL21-1gM-ELISA （90.3% and 87.0%）, and rOmpLI-lgM-ELISA （85.6% and 81.1%） （P〈0.01）. All IgM-ELISAs tested negative against 56 non-leptospirosis patients with typhoid fever, hemorrhagic fever or dengue fever. Conclusion Trigeminal fusion antigen increases ELISA sensitivity and the rLipL32/1-LipL21-OmpL1/2- IgM-ELISA is a sensitive and specific serological diagnostic method for clinical leptospirosis.

Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

Objectives To construct a recombinant plasmid carrying enhanced green fluore- scent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expre- ssion in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP- C1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluoresce- nce microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection. Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

汉坦病毒GM04-38株包膜糖蛋白基因表达载体的构建及表达

目的构建汉坦病毒GM04-38株包膜糖蛋白G1、G2的真核表达载体并将其在Vero E6细胞中进行表达,观察细胞融合现象。方法采用PCR和基因克隆技术,将GM04-38株G1、G2基因分别克隆到表达载体pCAGGS/MCS,酶切和测序鉴定正确后,将表达载体pCAGGS-G1、pCAGGS-G2共转染Vero E6细胞,酸性MEM处理后观察细胞融合现象的产生,并以间接免疫荧光试验(IFA)观察糖蛋白的表达情况。结果显示在Vero E6细胞中成功表达出糖蛋白G1、G2,IFA显示荧光信号分布在细胞浆中。二者的共表达可产生良好的生物学活性,在偏酸性条件下引起Vero E6细胞发生融合,而转染pCAGGS-NP的细胞没有融合现象发生。共表达也未出现明显的促进效应。结论成功构建了糖蛋白G1、G2的表达载体,并在Vero E6细胞中呈有效表达,二者的共表达可在偏酸性条件下引起Vero E6细胞发生融合。

Optimization of the Purification Methods for Recovery of Recombinant Growth Hormone from Paralichthys olivaceus

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone （r-fGH） was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield （2.605 mg L^-1） than that from soluble protein （1.964 mg L^-l）. Of the tested recovery methods, addition of renaturing buffer （pH 8.5） into denatured inclusion body yielded the best recovery rate （17.9%）. This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

Production of Rabbit Polyclonal Antibody Using hAPOA1 Protein Expressed in Prokaryotic Cells

The open reading frame （ORF） of hAPOA1 was inserted into the prokaryotic expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX- 4T-I-hAPOA1, which was then transformed into Escherichia coil strain BL21. The expression of target fusion protein was induced with isopropyl β-D-l-thiogalacto- pyranoside （IPTG）. The purified fusion protein in inclusion bodies was used to immunize New Zealand white rabbits to prepare hAPOA1 antiserum and the antibody titer was detected with indirect enzyme-linked immunosorbent assay （ID-EL1SA）. ID-ELISA and Western Blot proved that rabbit polyclonal antibody with a high titer of 1 ： 40 000 was produced, which may bring considerable economic benefits.

刍议具象表现主义油画与中国传统绘画精神的融合

具象表现主义和中国传统绘画有着许多共同的表征,在中国发展有其适宜的土壤,因此,对于中国具象表现主义油画家来说,当油画不再拘泥于技巧而强调思想内涵,谋求自己绘画语言和风格的时候,从传统中寻找作品新的灵感也就成了必然的选择,艺术家自觉地借鉴中国传统绘画精神,将西方的"表现"同中国传统美学中的"意象"进行融合,取得了不俗的建树。

Engineering Saccharomyces cerevisiae for enhanced(–)-α-bisabolol production

(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharma-ceuticals.However,the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement.This study aimed to develop a sustainable strategy to enhance the biosynthesis of(–)-α-bisabolol by metabolic engineering.By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9,a de novo(–)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L(–)-α-bisabolol.Two key genes for(–)-α-bisabolol biosynthesis,ERG20 and MrBBS,were fused by a flexible linker(GGGS)3 under the GAL7 promoter control,and the titer was increased by 2.9-fold.Optimization of the mevalonic acid pathway and multi-copy integration further increased(–)-α-bisabolol production.To promote product efflux,overexpression of PDR15 led to an increase in extracellular production.Combined with the optimal strategy,(–)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L,which is the highest titer reported in yeast to date.This work provides a reference for the efficient production of(–)-α-bisabolol in yeast.

一种基于在线模型匹配与更新的人脸三维表情运动跟踪算法

提出一种基于在线模型匹配与更新的人脸三维表情运动跟踪算法.利用自适应的统计观测模型建立在线模型,自适应的状态转移模型结合改进的粒子滤波同时进行确定性搜索和随机化搜索,并且融合目标的多种测量信息减少光照和个体相关性的影响.利用所提出的算法既可以得到全局刚体运动参数,又可以得到局部柔性表情参数.实验证明了该算法的有效性.