Cloning and Prokaryotic Expression of P23 Major Surface Protein Gene from Theileria sergenti

[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.

Cloning and Prokaryotic Expression of FnBP Ligand Binding Gene of Staphylococcus aureus

[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.

Cloning and Prokaryotic Expression of NS1 Gene of Porcine Parvovirus (PPV) SD1 Strain

[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a （ ＋ ） with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.

Molecular Cloning, Sequence Analysis and Prokaryotic Expression of Ovine Activin Receptor Type IIB(ActRIIB) Gene

Objective] This study aimed to clone ovine activin receptor type llB （Ac-tRIIB） gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction （PCR） using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 （DE3）. The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length （Genbank accession number： JX422071.1） with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology （99.6%） with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function.

Construction of Prokaryotic Expression Vectors of EBP1 Gene from Nervilia Fordii (Hance) Schltr.

[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.

Cloning, Analysis and Prokaryotic Expression of DsSP Gene from Dunaliella salina

[Objective] The purpose of this study was to clone a starch phosphorylase gene from Dunaliella salina and to preliminarily analyze its basic properties and protein expression. [Method] RT-PCR and RACE （rapid amplification of cDNA ends） method was used for gene cloning; basic properties of the gene were analyzed using bioinformatics method; prokaryotic expression vector PGS21a-DsSP was constructed and transformed into E. coil BL21; the fusion protein was purified and detected by GST-SefinoseTM Kit and Western Blot, respectively. [Result] A starch phos-phorylase gene （GenBank accession No. KF061044） named DsSP was successfully isolated from D. salina. Basic properties, subcellular localization, secondary structure and tertiary structure of the protein were analyzed and predicted. The fusion protein was found in the supernatant and inclusion bodies. The supernatant protein was successfully purified. Western Blot analysis showed that the fusion protein was successfully expressed in E. coil BL21. [Conclusion] This study laid experimental foun- dation for further clarifying the function and mechanism of DsSP.

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.

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.

Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.

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.

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.

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.

cDNA Cloning, Prokaryotic Expression of Two Splicing Products of mLRG, a Mouse Gene of Lipopolysaccharide Response

Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipopolysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns;mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.

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.

Prokaryotic Expression of P1 Gene of Type Asia1 Foot and Mouth Disease Virus(FMDV)and the Preparation of Its Antiserum

[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gene cloning techniques,and then cloned into pET-32a（＋）plasmid;subsequently the recombinant plasmid was transformed into E.coli BL21（DE3）;after the IPTG induction and protein purification,SDS-PAGE analysis was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result]The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion]This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.

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.

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.

Prokaryotic Expression of Rice Ospgip1 Gene and Bioinformatic Analysis of Encoded Product

Using the reference sequences of pgip genes in GenBank,a fragment of 930 bp covering the open reading frame(ORF) of rice Ospgip1(Oryza sativa polygalacturonase-inhibiting protein 1) was amplified.The prokaryotic expression product of the gene inhibited the growth of Rhizoctonia solani,the causal agent of rice sheath blight,and reduced its polygalacturonase activity.Bioinformatic analysis showed that OsPGIP1 is a hydrophobic protein with a molecular weight of 32.8 kDa and an isoelectric point(pI) of 7.26.The protein is mainly located in the cell wall of rice,and its signal peptide cleavage site is located between the 17th and 18th amino acids.There are four cysteines in both the N-and C-termini of the deduced protein,which can form three disulfide bonds(between the 56th and 63rd,the 278th and 298th,and the 300th and 308th amino acids).The protein has a typical leucine-rich repeat(LRR) domain,and its secondary structure comprises α-helices,β-sheets and irregular coils.Compared with polygalacturonase-inhibiting proteins(PGIPs) from other plants,the 7th LRR is absent in OsPGIP1.The nine LRRs could form a cleft that might associate with proteins from pathogenic fungi,such as polygalacturonase.

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.