Construction of eukaryotic expression vector of hypoxic inducible VEGF and its in vitro delivery application and functional identification / 解放军医学杂志

Objective To construct the eukaryotic expression vector of hypoxia inducible vascular endothelial growth factor (VEGF), and establish its in vitro delivery method. Methods Erythropoietin (EPO) enhancer was inserted into eukaryotic expression vector pGL4.73 [hRluc/SV40] (pSV) promoter by gene recombination technique to construct hypoxia inducible expression system (pEPO-SV). Renilla luciferase (Rluc) was used as downstream reporter gene. Then the VEGF165 gene was inserted into the pEPO-SV plasmid instead of Rluc, and the pEPO-SV-VEGF and pSV-VEGF expression vectors were obtained by inserting the pEPO-SV-VEGF gene into pSV as control. The pSV plasmid expressing Rluc or VEGF165 and pEPO-SV plasmid were transfected in vitro into human embryonic kidney 293T cells. The expression of Rluc or VEGF165 was used to identify the hypoxia induction function of the constructed vector after being treated under normal and hypoxic conditions for 24h and 48h. The intracellular delivery method of plasmids was then established based on poly (lactic acid-glycolic acid) copolymer (PLGA) nanoparticles as carrier, and the efficiency of the eukaryotic expression plasmids induced by hypoxia was evaluated under the in vitro hypoxia model. Results In the construction of plasmid, the successful insertion and correctness of EPO enhancer and VEGF165 gene were confirmed by restriction endonuclease digestion, PCR amplification and DNA sequencing. The plasmid expressing Rluc or VEGF165 was transfected into 293T cells respectively. There was no significant difference in the expression of reporter gene Rluc (one, plasmid pSV and pEPO-SV fluorescence expression values were 2448.24±158.51 and 3173.97±379.92, the second, plasmid pSV and pEPO-SV fluorescence expression values were 55 500.00±3237.05 and 51 193.18±866.32, respectively) or target gene VEGF165 in normal culture (P>0.05). But the expression of Rluc (In the cobalt chloride of hypoxia, the fluorescence expression values of pSV and pEPO-SV were 4857.70±1223.28 and 16 432.64±1618.73, respectively. In the hypoxia incubator, the fluorescence expression values of pSV and pEPO-SV were 2504.45±213.20 and 17 274.35±685.60, respectively) or VEGF165 in hypoxia was significantly higher than that in control group (P<0.01). The results showed that the constructed pEPO-SV and pEPO-SV-VEGF plasmids had typical hypoxia inducible expression activity. PLGA nanoparticles were used to in vitro deliver pEPO-SV and pSV in 293T cells. The results of detecting the reporter gene Rluc in normal culture and hypoxic conditions were consistent with those mentioned above, that is, under normal conditions, the 24h and 48h fluorescence expression values of plasmids pSV and pEPO-SV were 149.44±4.01 and 127.09±15.05, 1074.91±114.78 and 1064.56±137.48, respectively; under hypoxic conditions, the 24h and 48h fluorescence expression values of pSV and pEPO-SV were 3265.34±440.00 and 8828.87±637.03, 3202.06±33.43 and 9114.75±292.06, respectively. Conclusion A typical hypoxia inducible VEGF eukaryotic expression system has been successfully established, and an in vitro effective delivery method is also established, which may have an important application prospect in ischemia, hypoxia and other tissue injury diseases.

Advances & challenges in leptospiral vaccine development

Considerable progress has been made in the field of leptospiral vaccines development since its first use as a killed vaccine in guinea pigs. Despite the fact that the immunity conferred is restricted to serovars with closely related lipopolysaccharide antigen, certain vaccines have remained useful, especially in endemic regions, for the protection of high-risk individuals. Other conventional vaccines such as the live-attenuated vaccine and lipopolysaccharide (LPS) vaccine have not gained popularity due to the reactive response that follows their administration and the lack of understanding of the pathogenesis of leptospirosis. With the recent breakthrough and availability of complete genome sequences of Leptospira, development of novel vaccine including recombinant protein vaccine using reverse vaccinology approaches has yielded encouraging results. However, factors hindering the development of effective leptospiral vaccines include variation in serovar distribution from region to region, establishment of renal carrier status following vaccination and determination of the dose and endpoint titres acceptable as definitive indicators of protective immunity. In this review, advancements and progress made in LPS-based vaccines, killed- and live-attenuated vaccines, recombinant peptide vaccines and DNA vaccines against leptospirosis are highlighted.

Molecular differences between recombinant coagulation factors and plasma-derived coagulation factors, and their effects on pharmacology and efficacy / 国际药学研究杂志

Since their successful launch, the recombinant coagulation factors have been effective in the treatment and prophylaxis of respective deficiencies, and started to replace plasma-derived coagulation factors. However, immunogenicity and induction of inhibitors associated with the uses of these recombinant products have drawn certain attention. This manuscript reviews the structural differences between several major recombinant coagulation factors and their plasma-derived counteiparts, including sequences and many forms of post-translational modifications, and discusses the possible effects of these structural differences on the pharmacology, efficacy and pharmacokinetics of the products. The demonstration of structural differences and underlying mechanisms helps to improve the production yield and quality of recombinant coagulation factors, which is of significant clinical importance.

Molecular differences between recombinant coagulation factors and plasma-derived coagulation factors, and their effects on pharmacology and efficacy / 国际药学研究杂志

Since their successful launch, the recombinant coagulation factors have been effective in the treatment and prophylaxis of respective deficiencies, and started to replace plasma-derived coagulation factors. However, immunogenicity and induction of inhibitors associated with the uses of these recombinant products have drawn certain attention. This manuscript reviews the structural differences between several major recombinant coagulation factors and their plasma-derived counterparts, including sequences and many forms of post-translational modifications, and discusses the possible effects of these structural differences on the pharmacology, efficacy and pharmacokinetics of the products. The demonstration of structural differences and underlying mechanisms helps to improve the production yield and quality of recombinant coagulation factors, which is of significant clinical importance.