RESUMO
<p><b>OBJECTIVE</b>To construct an eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and obtain gp120 gene expression in HepG2 cells.</p><p><b>METHODS</b>According to the published gp120 gene sequence in Genbank, a pair of primers was designed and synthesized. The PCR amplification product of gp120 gene was cloned into pMD-18T vector using TA cloning followed by BamHI and XhoI digestion and sequence analysis. The target gene was then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid was sequenced and identified by restrictive endonuclease digestion, and transfected into HepG2 cells via liposome. The expression of gp120 gene was analyzed by RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Restriction endonuclease digestion and sequence analysis verified successful construction of the recombinant vector pcDNA3.1(+)/gp120. The target fragment gp120 was identical with U26942 in Genbank, and the expression of gp120 gene was detected in the lysate of the transfected HepG2 cells by RT-PCR and Western blotting.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmid for gp120 has been constructed successfully, which is capable of stable expression in HepG2 cells.</p>
Assuntos
Humanos , Vacinas contra a AIDS , Genética , Sequência de Bases , Carcinoma Hepatocelular , Genética , Metabolismo , Patologia , Linhagem Celular Tumoral , Clonagem Molecular , Células Eucarióticas , Metabolismo , Expressão Gênica , Proteína gp120 do Envelope de HIV , Genética , HIV-1 , Genética , Neoplasias Hepáticas , Genética , Metabolismo , Patologia , Dados de Sequência Molecular , Plasmídeos , Genética , Transfecção , Vacinas de DNA , GenéticaRESUMO
The viral spike protein is the main surface antigen of the coronavirus, and it could be useful in the research of clinical diagnosis, SARS vaccine and the structure biology.According to the analysis of the main antigen of the SARS spike protein, 5 fragments of the whole spike gene were cloned, and ligated to the vector pNMT1. Through electroporation transformantion to TCP1, the recombinant S. pombe strains capable of expressing the 5 fragments were constructed. SDS-PAGE or Western blot analysis of the induced expression products demonstrated that the 5 recombinant proteins were expressed in the fission yeast respectively.
Assuntos
Clonagem Molecular , Eletroporação , Glicoproteínas de Membrana , Genética , Proteínas Recombinantes , Genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Genética , Schizosaccharomyces , Genética , Metabolismo , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral , GenéticaRESUMO
The Cry1Ab differs most significantly from the other related ICPs by its absence of a carboxyl terminus of 28 amino acids including four cysteines; consequently it is less stable. We report that the helper protein P20 plays a role in the expression and crystallization of Cry1Ab. Three Cry1Ab expression plasmids pT1B, pP1B, and pDP1B, were constructed based on the shuttle vector pHT3101. The vector pT1B does not contain the p20 gene, pP1B carries p20, and pDP1B contains p20 with cry1A(c) promoter. Transformants were obtained by electroporating the plasmids into Bacillus thuringiensis acrystalliferous mutant CryB. Western blot demonstrated that crylAb was expressed as a 130 kD protein in all the transformants, and some of the protein was partially degraded into a 60 kD peptide. Quantitative protein analysis indicated that the amount of the 130 kD protein varied in the transformants and was in the ratio of 1:1.4:1.5 for PT1B, pP1B and pDP1B respectively. For the 60 kD proteins, the ratio was 1:1.1:1.6. Microscopic examination revealed that the size of the typical pyramidal crystals in the three transformants was in the order of T1B < P1B < DP1B. Bioassay showed that T1B, P1B and DP1B were all toxic to the larvae of Helicoverpa armigera with similar LC50. This study suggested that P20 plays a role in the expression and crystallization of Cry1Ab.
Assuntos
Animais , Bacillus thuringiensis , Genética , Metabolismo , Proteínas de Bactérias , Genética , Metabolismo , Farmacologia , Bioensaio , Métodos , Western Blotting , Eletroporação , Endotoxinas , Genética , Metabolismo , Farmacologia , Proteínas Hemolisinas , Genética , Metabolismo , Farmacologia , Microscopia Eletrônica de Transmissão , Mariposas , Regiões Promotoras Genéticas , GenéticaRESUMO
The vip3 A gene in a size of 2.3 kb amplified from wild-type Bacillus thuringiensis strain S184 by PCR was cloned into pGEM-T Easy vector and its sequence was analysized by DNASTAR. The plasmid pOTP was constructed by inserting vip3A-S184 gene into the expression vector pQE30 and then was transformed into E. coli M15. E. coli M15 cells harbouring the plasmid pOTP were induced with 1 mmol/L IPTG to express 89 kD protein which was confirmed to be Vip3A-S184 by Western blot. Experiments showed that about 19% of Vip3A-S184 proteins were soluble, and others were insoluble proteins and formed inclusion bodies observed by transmission electron microscopy(TEM). The target protein was purified under the native condition and the polyclonal antibody was prepared by immunizing rabbits. The polyclonal antibody was used to detect Vip3A proteins expressed in Bacillus thuringiensis. Bioassay showed that Vip3A-S184 showed a high toxicity against 3 tested insect larvae including Spodoptera exigua, Spodoptera litura and Helicoverpa armigera.