RESUMO
Lecithin cholesterol acyltransferase (LCAT) is the major enzyme producing most plasma cholesterol esters(CE) and a key participant in the process of reverse cholesterol transfer (RCT). The aim of this research is to co-express LCAT and it's natural activator apoA-I, with the recombinant adeno-associated virus vectors in the skeletal muscle cells, in order to pave a new way for gene therapy of the primary or secondary LCAT deficiency. 293T cells was cotransfected with pDG and rAAVAIL/rAAVL plasmids to produce infectious rAAV, and non-ionic iodixanol gradient centrifugation, followed by heparin affinity chromatography, were performed for separation, purification and concentration of rAAV. The particle numbers of rAAV, assayed by dot blot, were 7 x 10(14)/L (rAAVAIL) and 1 x 10(14)/L (rAAVL). These vectors were then transduced into C2C12 myoblasts. The results of ELISA and Western blot for human apoA-I, and [3H]-cholesterol-labeled radiochemical methods for LCAT activity, showed that the expression of human apoA-I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 30 days, even after myoblasts were differentiated into myotubes. PCR products for the transgene indicated the long-term persistence of transduced vector sequences. The results indicate that the methods used for production and purification of rAAV is efficient, and rAAV vector mediated the expression and secretion of LCAT and apoA-I gene in C2C12 myoblasts successfully. It suggests that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/or apoA-I cDNA in skeletal muscle in vivo can be a safe and fesible strategy for the gene therapy of LCAT deficiency.
Assuntos
Apolipoproteína A-I/metabolismo , Músculo Esquelético/enzimologia , Fosfatidilcolina-Esterol O-Aciltransferase/biossíntese , Animais , Western Blotting , Células Cultivadas , Dependovirus/genética , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Humanos , Camundongos , Músculo Esquelético/citologia , Fosfatidilcolina-Esterol O-Aciltransferase/genética , TransfecçãoRESUMO
AIM: To explore the regulatory effect of deltaN IkappaBalpha gene on the activity of nuclear factor-kappaB(NF-kappaB). METHODS: Ser32-and Ser36-deleted IkappaBalpha gene (deltaN IkappaBalpha) was cloned into adenovirus vector, and a replication-defective recombinant deltaN IkappaBalpha adenovirus(Ad-deltaN IkappaBalpha) was generated. A549 cells were divided into three groups: LPS-stimulated groups, Ad-LacZ+LPS group and Ad-deltaN IkappaBalpha+LPS group. Ad-LacZ+LPS group and Ad-deltaN IkappaBalpha+LPS group were infected with Ad-LacZ and Ad-deltaN IkappaBalpha, respectively, two days before LPS stimulation. The NF-kappaB activity of A549 cells was detected by Western blot and electrophoretic mobility shift assay (EMSA). TNF-alpha and IL-6 in the culture supernatant were detecteded by ELISA. RESULTS: The activity of NF-kappaB and the levels of TNF-alpha and IL-6 from Ad-deltaN IkappaBalpha virus-infected A549 cells were significantly decreased as compared with that of LPS-stimulated group and Ad-LacZ+LPS group. CONCLUSION: The results indicated that deltaN IkappaBalpha may inhibit the activation of NF-kappaB and reduce the release of TNF-alpha and IL-6, suggesting the recombinant deltaN IkappaBalpha adenovirus may be used for anti-inflammatory therapy.
Assuntos
Adenoviridae/genética , DNA Recombinante/genética , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Deleção de Sequência , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND & OBJECTIVE: The aim of this study was to express and purify human endostatin and to prepare polyclonal antibody of mouse anti-human endostatin. METHODS: The cDNA of endostatin was amplified by PCR, then recombined into prokaryotic expression vector and transformed into Escherichia coli BL21 for expression; the mice were immunized with purified products. RESULTS: Prokaryotic expression vector pQE-30 of human endostatin was successfully constructed; the expression product was gained after pQE-30 was transferred into BL21. After purified by Ni affinity chromatography, the product was identified to be a single component by SDS-PAGE. Western blot analysis showed that high titer mouse anti-human endostatin polyclonal antibody was successfully prepared. CONCLUSION: Highly purified expression product and prepared polyclonal antibody provide the necessary material for further study.