RESUMEN
<p><b>OBJECTIVE</b>To evaluate the expression of recombinant plasmid pcDNA3.1/GBD of glucan binding protein of Streptococcus mutans in mammalian cells COS-7.</p><p><b>METHODS</b>Eukaryotic plasmid carrying encoding gene of GBD of Streptococcus mutans gbpA was constructed and the plasmid was introduced into COS-7 cells by Lipofectamine reagent. The transient expressed protein in COS-7 cells was detected by immunochemistry technique.</p><p><b>RESULTS</b>The positive expression was detected in plasma of the cells which were transfected with recombinant plasmid pcDNA3.1/GBD. The cells which were transfected with pcDNA3.1 were negative.</p><p><b>CONCLUSION</b>GBD can translate and express in COS-7 cells after transfected with recombinant plasmid pcDNA3.1/GBD. The expressed protein locates in the plasma and the protein is able to combine with anti-GbpA antibody. The expressed protein has the antigenicity and is a candidate gene vaccine.</p>
Asunto(s)
Animales , Humanos , Antígenos de Superficie , Genética , Alergia e Inmunología , Proteínas Bacterianas , Genética , Alergia e Inmunología , Células COS , Proteínas Portadoras , Genética , Alergia e Inmunología , Caries Dental , Células Eucariotas , Metabolismo , Expresión Génica , Vectores Genéticos , Lectinas , Mamíferos , Plásmidos , Genética , Alergia e Inmunología , Proteínas Recombinantes , Streptococcus mutans , Genética , Metabolismo , Transfección , Vacunas de ADNRESUMEN
To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.