Higher expression level and lower toxicity of genetically spliced rotavirus NSP4 in comparison to the full-length protein in E. coli

Background: Rotavirus group A [RVA] is recognized as a major cause of severe gastroenteritis in children and new-born animals. Nonstructural protein 4 [NSP4] is responsible for the enterotoxic activity of these viruses in the villus epithelial cells. Amino acids 114-135 of NSP4 are known to form the diarrhea-inducing region of this viral enterotoxin. Therefore, developing an NSP4 lacking the enterotoxin domain could result in the introduction of a new subunit vaccine against rotaviruses in both humans and animals

Objectives: The aim of this study is the evaluation of rotavirus ANSP4 expression in E. coli expression system before and after removal of the diarrhea-inducing domain, which is the first step towards further immunological studies of the resulting protein

Materials and Methods: Splicing by overlap extension [SOEing] PCR was used to remove the diarrhea-inducing sequence from the NSP4 cDNA. Both the full-length [FL-NSP4] and the spliced [S-NSP4] cDNA amplicons were cloned into pET-32c and pGEX-6P-2. Expression levels of the recombinant proteins were evaluated in E. coli BL21 [DE3] by Western blot analysis. In addition, the toxicity of pET plasmids bearing the S-NSP4 and FL-NSP4 fragments was investigated by plasmid stability test

Results: For FL-NSP4, protein expression was detected for the strain containing the pGEX:FL-NSP4 plasmid, but not for the strain carrying pET:FL-NSP4. Hourly sampling up to 3 h showed that the protein production decreased by time. In contrast, expression of S-NSP4 was detected for pET:S-NSP4 strain, but not for pGEX:S-NSP4. Plasmid stability test showed that pET:S-NSP4 recombinant plasmid was almost stable, while pET:FL-NSP4 was unstable

Conclusions: This is the first report of production of rotavirus NSP4 lacking the diarrhea-inducing domain [S-NSP4]. SNSP4 shows less toxicity in this expression system and potentially could be a promising goal for rotavirus immunological and vaccine studies in the future

Comparison of rotavirus rf strain and hsv-1 titration by CCID50% and plaque assays

Titration of viruses is important to determine the quantity of virus in vaccine development, master virus seed stock preparation, viral vector studies and virus replication. In this study, we compared the CCID50% and plaque assay as a standard titration method for rotavirus [RF] and HSV-1. The MA104 and Vero cells were inoculated by RF and HSV-1 in 6- and 96- well plates. Following infection and adsorption, the optimal time for the cytopathic effect caused by the viruses was noted and the results compared. The CPE[Cytopathic Effect] of RF was observed in less than 18 hours, which increased until 72 hours after inoculation. In HSV-1, the CPE was observed 24 and 72 hours after inoculation. The virus titration in the plaque assay was monitored at 96 hours post-infection for RF and at 72 hours post-infection for HSV-1. In both viruses the plaque titer method was lower than the CCID50 method, since the results indicated that 1 CCID50% was equal to 0.7 PFU. The plaque assay is one of the most accurate methods for viral titration. For the plaque assay, individual lesions may be isolated, which the plaques can be counted. The CCID 50% method is not applicable for purification of homogenous viruses, nor is this technique reproducible

Immunogenicity of a new HIV-1 DNA Construct in a BALB/c mouse model

Cell mediated immunity, especially cytotoxic T cell responses against HIV-1 infection, plays a critical role in controlling viral replication and disease progression. DNA vaccine is a novel technology which is known to stimulate strong cellular immune responses. Many DNA vaccines have been tested for HIV infection but there is still no effective vaccine against this infection. Construction of a vaccine consisting of multiple conserved and immunogenic epitopes may increase vaccine efficacy. In the present study a DNA vaccine candidate constructed from HIV-1 P24-Nef was evaluated and cellular immune responses were assessed in murine BALB/c model. HIV-1 P24-Nef gene was cloned in PCDNA3.1 expression vector. Mice were immunized with DNA construct and IL-4 and IFN-gamma evaluation was per-formed using ELISPOT. Cytotoxicity response was evaluated with Granzyme B ELIS-POT assay and lymphocyte proliferation was evaluated with LTT assay. Analysis of immune responses showed that, compared to control groups, the candidate vaccine induced production of higher levels of both IL-4 and IFN-gamma [p<0.05]. Cytotoxicity and lymphocyte proliferation responses of mice vaccinated with the candidate vaccine were significantly increased compared to control groups [p<0.05]. HIV-1 P24-Nef DNA construct displayed strong immunogenicity in a murine model