Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Lipoxygenase （LOX, EC1.13.11.12） is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a（＋） and transformed into Escherichia coil BL21 （DE3）. Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside （IPTG） and the purified recombinant protein was obtained by His.Bind Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 （acetate buffer） and the optimum temperature was 30℃ for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

Construction of recombinant plasmid and prokaryotic expression in E. Coli and biological activity analysis of human placenta arresten gene

BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.

Prokaryotic Expression, Ascitic Polyclonal Antibody Preparation and Identification of Cashmere Goat Izumo1

Izumol is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumol was fractionated into 6 fragments （F0-F5） which were ligated with pGEX-4T1 to construct the prokaryotic expression vectors pGEX-Fn. The recombinant plasmids were transformed into Escherichia coli BL21 （DE3） and the GST-Fn fusion proteins were expressed successfully by induction with IPTG. GST-F0, a recombinant fusion protein of GST with the full length of extracellular region of mature cashmere goat Izumol, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumol ascetic polyclonal antibody with intraperitoneal injection of S 180 cells. Subsequently, the anti-GST-Izumol polyclonal antibody was purified with miscellaneous antigen by glutaraldehyde cross-linking method. Western blotting analysis showed that the purified ascetic polyclonal antibody had high affinity to all 6 GST-Izumol fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumol proteins were at the equatorial segment of sperm head surface. These results indicate that this polyclonal antibody has high specificity and lays the foundations for further study on the expression pattern of Izumol in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumol to the egg surface.

Prokaryotic Expression and Biological Activity Analysis of Human Arresten Gene

To express recombinant arresten in Escherichia coli (E.Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was amplified from recombinant plasmid pGEMArr by polymerase chain reaction (PCR), and inserted into prokaryotic expression vector pRSET containing T7 promoter. Restriction analysis and DNA sequencing verified that the arresten gene was correctly cloned into the expression vector. The recombinant plasmid pRSETAt was subsequently transformed into E.coli BL21 (DE3), and the target gene was expressed under induction of IPTG. SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 29 kD (1 kD=0.992 1 ku) amounted to 29 % of the total bacterial proteins. After purification and renaturation, the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of a biologically active form of human arresten in the pRSET expression system laid a foundation for further study on the mechanistic insight into arresten action on angiogenesis and the development of powerful anti-cancer drugs.

Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression

The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.

Prokaryotic Expression of Gene Encoding Glutamate Dehydrogenase of Streptococcus suis Serotype 2 and Preparation of Polyclonal Antibodies against Its Expressed Products

[ Objective] To obtain detection antigen for diagnosis of Streptococcus suis infection. [ Method] The complete ORF of glutamate dehy- drogenase （GDH） gene was amplified from the genomic DNA of Streptococcus suis serotype 2 strain SC22 isolated in Sichuan Province by poly- merase chain reaction （PCR）. The resulting product was cloned into the prokaryotic expression vector pET-30a, which was then transformed into E. coil BL21 （DE3）. The identified positive transformants were screened for expression induced by IPTG. The expression products were subjected to SDS-PAGE and the recombinant protein was purified by nickel ion-agarose affinity chromatography. New Zealand rabbits were immunized with the purified recombinant GDH protein to prepare polyclonal antibodies. Titers of the anti-serum were determined by indirect ELISA and Western blot assay. [ Result] The recombinant GDH protein was effectively expressed in the host bacteria, and highly pure recombinant protein was obtained by nickel ion-agarose affinity chromatography. High-titer anti-serum against the recombinant protein was obtained. As evidenced by western blot as- say, the sera could react specifically with the lysates of all detected Streptococcus suis strains. In addition, the recombinant GDH protein could re- act specifically with serum samples collected from five pigs experimentally infected by strain SC22. [ Conclusion] The expressed GDH fusion protein has some common epitopes of natural GDH and can be used as detection antigen to develop ELISA and other diagnostic methods.

Prokaryotic Expression,Purification,Antibody Production of a NH_2-Terminal Fragment of mCLCA3 Protein and Analysis of mClCA3 Protein Expression in Asthmatic Mouse Lung

mCLCA3 is a member of calcium activated chloride channel（CACC） family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E. coli, purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen, Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrate that the 90 kDa N-terminal peptide, neither the flail-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.

Construction of Prokaryotic Expression Plasmid of mtrC Protein of Neisseria gonorrhoeae and Its Expression in E.Coli

Summary： In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimierobial hydrophobie agents and study on the resistant mechanism of multiple transferable resistance （mtr） efflux system, plasmid pET-28a（＋） encoding mtrC gene was constructed and the related target protein was expressed in Escherichia colt （E. cold DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a（＋） with restriction endonuelease to construct recombinant pET-mtrC which was verified by restriction endonuelease and DNA sequencing. The recom- binant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuelease and DNA sequencing. hs sequence was 99.5 % homologus to that published on GeneBank （U14993）. A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli.

Construction of Prokaryotic Expression Vector for pbv220/NT4-ADNF-9

Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 cDNA was synthesized using asymmetrical primer/ templates and ligated to the 3’ terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4 -ADNF-9, was subcloned into prokaryotic expression vector pBV220, and named pBV220/ NT4-ADNF-9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS-PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. Results The correct sequence of fusion gene NT4-ADNF-9 was successfully subcloned into the pBV220 vector. The expressed ADNF-9 protein showed its effects in promoting cell survival and neurite growth. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.

Cloning, Sequence Analysis, and Prokaryotic Expression of the Porcine DECR1 Gene

2,4-dienoyl-CoA reductase 1 （ DECR1 ） is the key rate-limiting enzyme in the metabolism of polyunsaturated fatty acids. Although this protein has been studied in a variety of mammals, its role in por- cine is yet to be fully elucidated. However, it is a candidate determinant/indicator of meat quality, growth traits, and carcass quality. Here, we employed RT-PCR and rapid amplification of cDNA ends （RACE） analysis to amplify the full-length cDNA of DECR1 from Mashen pig liver, and cloned it into the expression vector pET-32a＋. After confirmation by sequencing and restriction analysis, the recombinant plasmid was transformed into E. coli BL21 cells. The cDNA of pig DECR1 contained 2,352 nucleotides, including a 987 bp open reading frame flanked by a 53 bp 5＇-untranslated region （UTR） and a 1,312 bp 3＇-UTR. The pig DECR1 coding sequence encoded 328 amino acid residues, which shared 99%, 88%, 87%, 87%, 87%, 87%, and 83% identity with those of Sus scrofa （predicted）, Bos taurus, Homo sapiens, Macaca mulatta, Pan troglodytes, Equus caballus, Canis, and Mus musculus, respectively. SDS-PAGE analysis revealed that the recombinant protein was expressed and that the expression level reached its highest level after 4 h induction. Western blot analysis indicated that the molecular weight of the expressed protein was the same as that predicted, ap- proximately 35 kDa. Collectively these data provide the basis for further studies into the physiological functions and molecular mechanisms of the pig DE- CR1 gene.

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.

Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

Phosphatase of regenerating liver 3 （ PRL3 ）, which belongs to the superfamily of protein tyrosine phosphatases （PTPs）, represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes. Presented here are the results of cloning, prokaryotic expression, purification, and polyclonal antibody preparation of PRL3. To obtain a specific polyclonal antibody against PRL3, the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns. Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein. The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.

Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation

[Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip.

Phytoene Synthase Gene Cloning from Citrus sinensis Osbeck cv. Cara Cara and Its Prokaryotic Expression

Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.

Prokaryotic Expression and Identification of the WbkC Gene of Brucella melitensis

[ Objective] In order to clone and express the formyltransferase of Brucella abortus in E. coli, purify the expressed protein and detect its immunogenicity. EMethods] A gene recoding formyltransferase 27 -35 ku （Wbkc） was amplified from the genomic DNA of Brucella abortus PCR. The amplified fragments were digested with BamH I and Sal I, and then cloned to the vector pET28a. The constructed recombinant plasmid pET28a-Wbkc was transformed to E. coil BL21 and was induced to express the fusion protein. Then the protein was purified by histidine-binding res- in column chromatography, and the immunogenicity was detected by Western blot. The PCR product of Wbkc gene was cloned to the vector pET28a, and the recombinant vector was confirmed by colony PCR identification, recombinant vector digested identification and sequencing analy- sis. [ Results] It was successfully expressed in E. coil BL21 as a fusion protein with histidine at the presence of IPTG, and a specific protein band of 29 ku was found when identified by SDS-PAGE. Western blot showed good immunoreactivity of the expressed product. Wbkc was successfully cloned and expressed, and the purified fusion protein had immunogenicity. This study provides a solid foundation for the further study on the function of proteins and brucellosis diagnostic antigens. [ Conclusion] Amplification of formyltransferase gene of Brucella abortus and prokaryotic expression of WbkC_ indicatino the formvItransferase has specific immune reactivitv.

Prokaryotic Expression of Infectious Bronchitis Virus S1 Gene and Analysis of Biological Activity of Recombinant Protein

[Objective] To study the prokaryotic expression and antigenicity identification of S1 gene from avian infectious bronchitis virus （IBV）. [Method] The S1 gene was cloned into a pMD18-T vector to yield a recombinant plasmids pMD18-T-IBV-S1. Then S1 gene was inserted into the multiple cloning site of a prokaryotic expression vector pET-32a （ ＋ ）. The recombinant plasmid was transformed into E. coil BL21. The recombinant protein was induced by IPTG and measured by SDS-PAGE and western-blotting. [Result] The S1 gene was successfully expressed in E. coil BL21, the fusion proteins were about 66.0 kDa in a form of inclusion body. Western-blotting test showed that the recombinant proteins could be identified by IBV polyclonal antibody. [ Conclusion] The recombinant proteins of S1 gene have the antigenicity, which lays a good foundation for further research on new generation vaccine of IBV.

Construction and Expression of Prokaryotic Expression Vector of MPT-64 Gene

[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.

Cloning and Prokaryotic Expression of Canine IL-2 Gene

[ Objective] To clone and express canine IL-2 gene and thus to provide theoretical support for the development of novel immune enhancers and genetic engineering vaccines. [ Method] Leukocytes separated from canine whole blood were stimulated by concanavalin for 20 h, and then total RNA was extracted. According to the sequence of canine IL-2 gene published in the GenBank, a pair of primers was designed. After PCR am- plificetion, the target fragment was cloned into prokaryotic expression vector pET-28a. The recombinants were transformed into the host bacteria BL21. After IPTG induction, the expression products were analyzed by SDS-PAGE. [ Result] A 500 bp band with the expected size appeared in the RT-PCR products. After the pMD18-T-IL2 was identified by double digestion, an approximately 500 bp fragment was produced, which indicated successful cloning of the gene. After the pET-28a-lL2 was identified by restriction enzyme digestion and PCR, a 500 bp fragment was produced, which indicated successful construction of the expression vector. As revealed by the SDS-PAGE analysis, a protein band with molecular weight of about 20 kDa appeared. [ Conclusion] The canine IL-2 gene was cloned and expressed.

Prokaryotic Expression and Antigenic Analysis of Wbkc Gene from Brucella abortus

[ Objective ] This study aimed to clone and express formyltransferase （Wbkc） gene from Brucella abortus in E. coli, purify the expressed protein and analyze its immunogenicity. [Method] A gene encoding 27 -35 ku formyltransferase （Wbkc） was amplified from the genomic DNA of BruceUa abortus by PCR. The amplified fragments were digested with BamH I and Sal I, and inserted into pET28a vector. The constructed recombinant plasmid pET 28a-Wbkc was trans- formed into E. coli BL21 and was induced to express the fusion protein. Subsequently, the protein was purified by histidine-binding resin column chromatography, and the immunogenicity was detected by Western blot assay. The recombinant plasmid was identified by colony PCR, double digestion and sequencing analysis. [ Result] Wbkc was successfully cloned and expressed in E. coli. A specific protein band of 29 ku was detected by SDS-PAGE. Western blot showed specific im- munoreactivity of the purified fusion protein. [ Conclusion] This study provided a solid foundation for further investigating diagnostic proteins for brucellosis and developing Brucella gene-deletion vaccines.

Construction of Recombinant Expression Plasmids Containing H and F Protein Genes of Canine Distemper Virus Isolated from a Mink and Their Expression in Prokaryotic Cells

[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prekaryotic cells as well as to study the reactogenieity of the expressed products. [ Method ] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subclonlng techniques were used to construct the cloning plasmids（pMD-18T-H and pMD-18T-F） and recombinant expression plasmids（pET28a-H and pET28a-F） ; SDS-PAGE and Western-blotting were adopted to verify whether the target proteins were successfully expressed. [ Result] The recombinant expression plasmids pET28a-H and pET28a-F containing H and F protein genes of Canine distemper virus isolated from a mink were successfully constructed, and both the expressed H and F proteins with respectively relative molecular mass of 31 400 and 38 200 produced positive reac- tion with the CDV standard positive serum. [ Conclusion] The H and F proteins expressed in prokaryotic cells were the same with the natural ones in terms of reac- togenicity, which can be utilized for diagnosis of a CDV＇s infection or for an epidemiological investigation. Meanwhile, they also provide a basis for developing ge- netically engineered subunit vaccines.