Construction of Plant Expression Vector for Hand,Foot and Mouth Virus EV71-VP1 Gene and Its Expression in Tomato

EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.

The Construction and Identification of Eukaryotic Expression Vector pEGFP-N1-hTERT

[Objective] The aim of this study is to construct eukaryotic expression vector pEGFP-N1-hTERT and observe its expression in eukaryotic cells.[Method]The eukaryotic expression vector pEGFP-N1-hTERT was constructed with pC1-neo-hTERT and pEGFP-N1 plasmids,and the accuracy of human telomerase reverse transcriptase(hTERT)gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.After transfecting pEGFP-N1-hTERT into rat fetal neural stem cells(NSCs),the protein localization of human telomerase reverse transcriptase were indirectly observed through green fluorescent protein in the cells,and the correctness of constructed pEGFP-N1-hTERT was certificated by RT-PCR and Western Blot analysis.[Result]The eukaryotic expression vector pEGFP-N1-hTERT had correct structure and could express in eukaryotic cells.[Conclusion]This study laid a foundation for the establishment of immortalized NSCs line in rats.

Construction and Identification of a Goat Pox Virus Transfer Vector to Express Peste des Petits Ruminants H gene

[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vector; the recombinants were digested by Nhe Ⅰ and Hind Ⅲ, and ligated into pEGFP-N1-P7.5, yielding the recombinant vector pEGFP-N1-P7.5-H; next the expression cassette EGFP-N1-P7.5-H was first released from recombinant vector pEGFP-N1-P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ, yielding the transfer vector pUC119-TK-EGFP-P7.5-H. [Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7.5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h of transfection. [Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.

Construction analysis on integral lifting of steel structure by the vector form intrinsic finite element

A newnumerical method based on vector form intrinsic finite element（VFIFE） is proposed to simulate the integral lifting process of steel structures. First, in order to verify the validity of the VFIFE method, taking the steel gallery between the integrated building and the attached building of Nanjing M obile Communication Buildings for example, the static analysis was carried out and the corresponding results were compared with the results achieved by the traditional finite element method. Then, according to the characteristics of dynamic construction of steel structure integral lifting, the tension cable element was employed to simulate the behavior of dynamic construction. The VFIFE method avoids the iterative solution of the stiffness matrix and the singularity problems. Therefore, it is simple to simulate the complete process of steel structure lifting construction.Finally, by using the VFIFE, the displacement and internal force time history curves of the steel structures under different lifting speeds are obtained. The results show that the lifting speed has influence on the lifting force, the internal force, and the displacement of the structure. In the case of normal lifting speed, the dynamic magnification factor of 1. 5 is safe and reasonable for practical application.

Construction of a RNA Interference Vector of Brassica napus L.Pyruvate Kinase Gene

A predominantly expressed gene, pyruvate kinease （PK） gene, control ing oil accumulation, had been identified in previous study. To construct a PK RNAi vector, a 498-bp target PK gene sequence was amplified and transferred into the pEASY-T1vector. Subsequently, the target DNA fragments were cut off by enzymes Not I and Xho I and directional y inserted into plant RNAi platform vector pHurricane, a newly developed RNAi vector in our lab, to form the PK inverse repeats. The PK inverted repeats fragment was then cloned into a modified vector pCAMBIA1390, driven by a rapeseed seed-specific promoter napin. Restriction enzyme digestion verified the successful construction of RNA interference vector. The PK RNAi con-struction wil lay a foundation for study in the function of PK in oil accumulation and metabolism in rapeseeds.

Construction of Plant-based Expression Vector of Polyphosphate Kinase Gene

[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（L03719） of E.coli DH5α in Genbank.Genomic DNA of E.coli DH 5α was extracted as template for the amplification of PPK gene by PCR method.By using In-Fusion@ HD Cloning Kit,the PPK gene was directionally cloned into NcoI site of the pCAMBIA1302 vector.[Result] Sequencing results showed that the 2.0 kb long fragment of PPK gene was inserted into the plant-based expression vector pCAMBIA1302 in front of GFP gene.[Conclusion] The fusion expression vector of PPK and GFP genes were successfully constructed.

Construction of Plant Expression Vector for Recombinant Human Epidermal Growth Factor (hEGF)

ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccording to the hEGF sequence in GenBank, hEGF was synthesized artificially; subsequently, hEGF gene was ligated with green fluorescent protein （GFP） gene, and their ligation product was then amplified with primers flanked with corresponding endonuclease cleavage sites, followed by double digestion by Sal I and EcoR I of the amplified products; next, pRI 101 AN DNA was extracted and digested by both Sal I and EcoR I; susequently, the digestion products of hEGF and GFP ligation fragment by Sal I and EcoR I and the digestion products of pRI 101 AN plasmid DNA by Sal I and EcoR I were ligated, and their ligation product was transformed into Escherichia coli XL10-Gold, followed by extraction of DNA from the recombinants exhibiting green fluorescence, which was then identified by enzymatic digestion and PCR, and the verified recombinant plasmid DNA was named pBZG101. ResultHuman epidermal growth factor gene （hEGF） and green fluorescent protein gene （GFP） were successfully ligated, and their ligation fragment was successfully ligated to pRI 101 AN DNA, finally with the acquirement of the plant expression vector for recombinant human epidermal growth factor-（pBZG101）. ConclusionThe plant expression vector for recombinant human epidermal growth factor-（pBZG101）- was successfully constructed in this study.

Construction of Plant Virus Expression Vector pClYVV/CP/W and Expression of GFP

[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein. [ Method] A section of multiple cloning sites among NIb/CP genes in pCIYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease Nla were cloned with infectivity full-length cDNA of clover yellow vein virus （CIYVV）, and pCIYVV/CP/W vector was constructed, GFP gene was inserted into pCIyVV/CP/W to construct the pCIYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot （WB）. [Result] The broad bean seedling inoculated with pCIYVV/CP/W/GFP expressed the same symptom as wild type CIYVV, morbidity was of 100%, the result showed that recombinant virus clone pCIYVV/CP/W/GFP didn＇t suppress, insertion of foreign gene didn＇t destroy the open reading frame of pCIYVV/CP/W. Foreign gene can keep living in F, progeny virus genorne steadily, recombinant virus clone pCIYVV/CP/W/GFP could steadily express GFP in progeny virus at least.[ Conclusion] The useful plant virus vector was provided for useful protein expressing.

Cloning of Broad-spectrum Anti-disease NPR1 Gene with RT-PCR and Construction of Its Protein Expression Vector

[Objective] It is to clone broad-spectrum anti-disease gene NPR1 and to construct its protein expression vector.[Method] First to extract total RNA of Arabidopsis thaliana and design relevant primers,and then the method of reverse transcription PCR was adopted to clone.With the method of enzyme digestion and ligation,this gene will be directed into protein expression vector.[Result] After relevant testing,NPR1 was inserted into vector pMXB10 to obtain pMXB10-NPR1 protein expression vector.[Conclusion] Protein expression vector including NPR1 was successfully constructed.

Comparison of School Building Construction Costs Estimation Methods Using Regression Analysis, Neural Network, and Support Vector Machine

Accurate cost estimation at the early stage of a construction project is key factor in a project’s success. But it is difficult to quickly and accurately estimate construction costs at the planning stage, when drawings, documentation and the like are still incomplete. As such, various techniques have been applied to accurately estimate construction costs at an early stage, when project information is limited. While the various techniques have their pros and cons, there has been little effort made to determine the best technique in terms of cost estimating performance. The objective of this research is to compare the accuracy of three estimating techniques (regression analysis (RA), neural network (NN), and support vector machine techniques (SVM)) by performing estimations of construction costs. By comparing the accuracy of these techniques using historical cost data, it was found that NN model showed more accurate estimation results than the RA and SVM models. Consequently, it is determined that NN model is most suitable for estimating the cost of school building projects.

Cloning of a flower-specific expression promoter from Arabidopsis thaliana and its plant expression vector construction

Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank （AF248988）, a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter （AF248988） was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vectorpAtChs：：GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.

Expression Vector Construction and Genetic Transformation of <i>Rosa rugosa β</i>-l,3-Glucanase Gene (<i>RrGlu</i>)

In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.

Construction of Expression Vector for Porcine Gastrin-releasing Peptide Fusion Protein

[Objectives]This study was conducted to obtain porcine gastrin-releasing peptide(GRP)fusion protein.[Methods]The constructed pET32a(+)-GRP plasmid was transformed into Escherichia coli BL21(DE3)competent cells to obtain the pET32a(+)-GRP-BL21(DE3)fusion protein expression strain,which was induced with 0.5 mM IPTG at 25℃and 150 r/min for 12 h,and the His-tagged GRP fusion protein was detected by SDS-Page gel electrophoresis and Western Blot.[Results]After optimizing the IPTG-induced expression conditions,it was confirmed that the porcine GRP fusion protein was obtained,and the porcine GRP fusion protein was soluble,stable and highly active.[Conclusions]This study lays a foundation for the subsequent preparation of anti-pig GRP antibodies.

Construction of plant expression vector of Pseudopleuronectes americanus antifreeze protein gene

The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.

Construction of RNAi Expression Vector against Riboflavin Synthase Gene

[Objective] This study aimed to construct RNAi expression vector against r/boflavin synthase （RS） gene. [Method] By using the primers designed based on RS gone coding sequence that was screened from Arabidopsis cDNA library, the 476 bp cDNA fragment of RS was amplified from pGADTT-RS recombinant plasmid, and then cloned into pUCm-T vector to obtain pUCm-RS. Two RS fragments （476 bp） were obtained through digesting pUCm-RS with restriction enzymes PstI/BamHI and PstI / Xhol, and then respectively connected into vector pBSSK-in to form pBSSK-RS-in-RS, in which the two RS fragments were inverted re- peats. Finally, the transform unit RS-intron-RS, got by digesting vector pBSSK-RS-in-RS with Sac I and Kpn I, was ligated into expression vector pCAMBIA1301 to obtain the RS gene silencing vector. [ Result] The restriction enzyme digestion sequencing analysis proved that the RS gene silencing vector was successfully con- structed. [ Conclusion] This study may provide a basic material for further studies on the bio-function of RS gene and the mechanism of signal transduction induced by HpaGxoo in plant.

Construction of Prokaryotic Expression Vector for At4g12500 Gene of Arabidopsis

[ Objective] This study aimed to construct the prokaryotic expression vector for Arab/dops/s At4g12500 gene. [ Method] Genomic DNA of wild-type Ar- abidopsis Cold） was extracted as the template for amplification of At4g12.500 gene with PCR technology. The target fragment was double-digested with BamH I and Xho I, and ligated with prokaryotic expression vector pET-28a. [Result] Prokaryotic expression vector pEI28a-At4gI2500 was successfully constructed and trans- formed into Escherichia coli expression strain BL21 （DE3）. [ Conclusion] This study laid the foundation for subsequent expression and functional analysis of At4g12500 gene.