Human kerotinocyte growth factor (hKGF) gene amplified by PCR was inserted into the shuttle vector pAdTrack-CMV to get the recombinant plasmid pAdTrack-CMV-hKG, which was linearized with Pme I and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy-1 to obtain the recombinant adenoviral plasmid pAdEasy-hKGF. The recombinant adenoviral plasmid was then transfected into HEK-293 cell lines via Lipofectamine 2000 to package and amplify the recombinant adenovirus containing hKGF gene detected by PCR. The recombinant adenovirus produced could effectively infect HaCat cells. The result of Western blotting showed that HaCat cells infected with the recombinant adenovirus expressed and secreted hKGF protein.
Interferon-tau (IFN-tau) is a type I IFN originally discovered for its role as a pregnancy recognition hormone in ruminant animals such as sheep and cows. IFN-tau possesses all of the biological properties ascribed to the other type I IFNs including antiviral, antiproliferative and immunomodulatory activities. In order to identify the function of IFN-tau clearly, the coding sequence of IFN-tau was amplified by PCR from IFN-tau cDNA, this fragment digested by EcoR I and BamH I was inserted into pBV220 by T4 ligase and transformed into E.coli BL21 then the recombinant plasmid named pBV220/IFN-tau was constructed successfully. IFN-tau was expressed distinctly after inducing by the temperature. The expression production was purified on S-100 High Resolution. The product was identified as IFN-tau by amino acid sequence analysis. The specific activity of IFN-tau was about 2.09×106IU/ml assayed by the standard antiviral activity test on WISH cells challenged with VSV virus.
yggG, a Era-binding protein gene, was isolated and cloned from the E. coli genomic DNA library. Previous studies indicated that the product of yggG gene, YggG294(amino acids 1-294), strongly inhibited the growth of host bacteria and caused the death of bacteria cells. To elucidate the relationship between YggG and Era, we constructed a double promoter expression vector that can express YggG294 and Era proteins controllably in the same time. Using this vector to express YggG294 and Era protein in the same E. coli cells, then analyzed the relation between YggG294 and Era. The results showed that the ratio of Era proteins to total proteins increased with the increase of induction time in E. coli cells without YggG294 expression and with little YggG294 expression, while the ratio of Era proteins to total proteins seemed to be a constant level in E. coli cells overexpressing YggG294. The results of this study also showed that pre-expression of Era protein did not produce any effect on the growth inhibition of E. coli cells caused by YggG294. These results indicate that the expression of YggG294 produced a very strong inhibitory effect on the growth rate of E. coli cells and the overexpression of Era, ant that the interaction between YggG and Era is not associated with the growth inhibition of E. coli cells caused by YggG294.
Objective: To construct pET23d-HvSRP19 expression vector for the gene of SRP19 protein of halophilic archaeon Haloferax volcanii (Hv SRP19), and induce it to express in E.coli. Then to purify and analyze the biological activity of its expressed proteins. Methods: The Hv SRP19 full length gene sequences were firstly got from a set of 10 overlapping synthetic short oligonucleotides by de novo recombinant DNA techniques and splicing, and then cloned to pET23d vector. The large expressed products of recombinant plasmid in E.coli BL21(DE3)pLysS were purified by Q-Sepharose ion exchange chromatography, and its biological activity was then analyzed by sucrose density gradient ultra-centrifugation. Results: pET23d-HvSRP19 expression vector was correctly constructed, and found to have a very good expression in E.coli. The purification of the expressed products was confirmed to be successful with an achievement of the protein pure degree up to 95%, and the purified proteins were demonstrated to have the Hv SRP19 biological activity due to it could form complex with Hv SRP RNA. Conclusions：The availability of the Hv SRP19 interaction with Hv SRP RNA was considered to be the initiation of SRP formation and performing function.
In order to obtain a both cheap and high efficient SUMO protease ,a fusion gene Ulp1p , composed of 6xHis tag and Ulp1(Ubiquitin-like-specific protease 1)’s active fragment(403aa-621aa) was amplified by PCR. The Ulp1 was then cloned into pET3-c to form a expression plasmid pET - Ulp1p,which was transformed into BL21(DE3) subsequently screened by ampicillin. After induction by IPTG for 4h, the expression of fusion protein, Ulp1p,was detected by Abstract Recently, SUMO protease plays an important role in the purification of SUMO tag fusion protein expression system .In order to obtain a both cheap and high efficient SUMO protease ,a fusion gene Ulp1p , composed of 6xHis tag and Ulp1(Ubiquitin-like-specific protease 1)'s active fragment(403aa-621aa) was amplified by PCR. Importangtly ,the synthesized Ulp1p DNA sequence was designed on the basis of preferred codens of E. coli ,so it would help increase the expression of Ulp1p potentially in E. coli .The Ulp1p was then cloned into pET3-c to form a expression plasmid pET - Ulp1p,which was transformed into BL21(DE3) subsequently screened by ampicillin. After induction by IPTG for 4h, the expression of fusion protein, Ulp1p,was detected by 12%SDS-PAGE and Western blotting, The fusion prtein was up to 50.8% of total E. coli protein and expressed as supernatant. The fusion prtein was purified by Ni-NTA Resin chromatography.After passing G-25 to desalt, target protein was 12%SDS-PAGE pure. The catalytic reaction of Ulp1p to SUMO-hEGF(human epidermal growth factor) and GST-SUMO-MT(metallothionein) showed that the recombinant protein Ulp1p displays high specificity and activity. The specific activity of Ulp1p is 1.386 x104U/mg. Therefore, the high expression, specificity and activity of recombinant Ulp1p indicates the constructed genetic engineering E. coli BL21(DE3)/ pET - Ulp1p can be used for the large scale production of recombinant Sumo Protease Ulp1p.
The completed DNA of 1.7 kb HA gene was prepared from the cDNA by RT-PCR. The fragment was subcloned into the pFastBacHTa donor plasmids. the recombinant pFastBacHTa was cut by restriction enzyme and sequenced. then The purified plasmid DNA was transform into DH10Bac E. coli for transposition into the bacmid and the colonies was identified by blue/white selection. The recombinant bacmid was verified by PCR analysis. The recombinant baculovirus was transfected into sf9 cells by Cellfectin reagent, the expressed HA protein was analyzed by SDS-PAGE、Western-blot、HA test and HI test and it have good immunological activity.
Abstract：The purpose of this study was to compare the difference between transgene expressions driven by homologous duplicated carbonic anhydrase(DCA)promoter and foreign CaMV35S promoter in the unicellular green alga, Dunaliella Salina. We introduced the CaMV35S promoter-bar construct and DCA promoter-bar construct into D.Salina by a Backon 2000 electroporation system. After the repeated selection with the PPT of 3mg/L, 3 PPT-resistant phenotype transformants were isolated from the CaMV-bar and DCA-bar pools of transformants of D. Salina, respectively. The results of PCR and sequencing showed that bar genes were stablely integrated into the genome of D.Salina, and Sorthern bolt showed the number of transgene copy had no significant difference between both promoters. Semi-quantitive RT-PCR indicated that the mRNA levels of bar gene were higher in DCA-bar transformants than the CaMV-bar transformants, and can be increased with the induction of high salt in DCA-bar transformants while the CaMV-bar transformants showed no change with the salt induction. The analysis of growth rate of transformants showed DCA-bar transformants achieved the log stage faster than the CaMV-bar transformants. It is concluded that the homologous promoters have more advantages than the foreign promoter in the transgenic D.Salina.
Mitochondrion is the basic and important organelle in most of the eukaryocyte, It is the place of oxidative phosphorylation. The gene order of the different vertebrate shows that the gene of cytochrome b(Cytb)、cytochrome oxidase I（COⅠ）、cytochrome oxidase II（COⅡ）and cytochrome oxidase III（COⅢ）are the most consevative,The homology is the highest. The genovariation of ATPase6、ATPase8 and ND is more much.The mitochondrial cytochrome oxidase III(COⅢ) and its associated tRNA-Gly、ATPase6 genes of domestic duck were sequenced by PCR.The open reading frame of COⅢ gene contains 784bp nucleotides,The COⅢ gene of domestic duck shows a high degree of homology with that from the other poultry recorded in the GenBank and it has 90.6% homology with Aythya Americana. 89% with Branta canadensis、88.6% with Anser albifrons and 88.6% with Cygnus columbianus.The phylogenetic trees show that the relationships based on the homology is consistent with the morphological and taxonomic results.
The expressions of StAR (steroidogenic acute regulatory protein) mRNA in testes from 7、14、23 and 37 day-old piglets were studied by tissue in situ hybridization. The results indicated that in the testes of piglets, StARmRNA was expressed in Leydig cells of pig testes. The expression level of StARmRNA was lower in 7 days piglets but higher in 14、23、37 days. The results indicated that StAR gene played an important role in steroid biosynthesis.
Abstract: Bovine antimicrobial peptides Bac7 and Bac5 are linear cation micromolecular polypeptides, which play an important role in both innate and acquired immunity. In the present study, based on the gene sequences encoding mature bovine antimicrobial peptides bac7 and bac5 as registered in Genbank, the fragment of the fusion gene Bac7-Bac5 was synthesized and cloned into the prokaryotic expression vector pET32 (a+) to construct a recombinant expression vector, pET-B7-B5. The recombinant construct was then transformed into E coli BL21 (DE3) to over-express the recombinant protein B7-B5 (rB7-B5). The expressed rB7-B5 was localized in inclusion bodies and accounted for 36.6% of total bacterial protein. The molecular weight of rB7-B5 was 33kD, a figure consistent with the predicted value. Following purification by Ni affinity chromatography and stepwise renaturation by dialysis, rB7-B5 showed sound antimicrobial activity in porcine actinobacillus pleuropneumoniae and antibiotics-resistant E. coli. The present study provides basis for the research and development of novel antibacterial preparations.
