ABSTRACT
Background: Designing novel therapeutic agents has been a critical challenge for HIV disease
Materials and Methods: In current study a DNA sequence which was encoded the Tat protein was synthesized and inserted in pET 28 vector. Vector was cloned in BL21-DE3 E. coli and cultured in TB media. After protein expression, recombinant Tat protein was purified by NTA affinity chromatography. The Tat purified protein efficiency and confirmed by SDS-PAGE and Western blot, respectively. We were immunized the camel against HIV-1 Tat recombinant protein to made a camelid antibody library. Total RNA was extracted from camel lymphocytes and VHH fragments synthesized and amplified using RT-PCR and Nested- PCR methods by special primers
Results: The 350- 450 bp VHH gene fragment was produced by RT-PCR and Nested- PCR and extracted from agarose gel 1%. Then gel extraction was performed and pure fragments were inserted in HEN-4 vector by T4 DNA ligase
Conclusion: The library can be applied for biopanning and isolation of nanobody against HIV-1 Tat Protein. Nanobody small size may be a useful drug for treatment of HIV disease because give them the potency of the recognizing the cryptic epitopes of tat and neutralized the virus
ABSTRACT
Monitoring of influenza virus shedding and optimization of multiplicities of infection [MOI] is important in the investigation of a virus one step growth cycle and for obtaining a high yield of virus in vaccine development and conventional basic diagnostic methods. However, eluted infectious viruses may still be present immediately after virus inoculation and when cells are washed following virus cultivation which may lead to a false positive virus infectivity assay. In this experimental study, we investigated influenza virus progeny production in Madin-Darby canine kidney [MDCK] cells with five different MOI at determined time points. The results were analyzed by end point titration tests and immunofluorescence assay. Higher titers of eluted virus were observed following a high MOI inoculation of virus in cell culture. Most probably, this was the result of sialic acid residues from viral hemagglutin in proteins that were cleaved by neuraminidase glycoproteins on the surface of the influenza virus, which promoted viral spread from the host cell to the culture supernatant or during endocytosis, where viruses recycle to the cell surface by recycling endosomes which culminated in virus shedding without replication. We demonstrated that the pattern of influenza virus progeny production was dose-dependent and not uniform. This production was influenced by several factors, particularly MOI. Understanding the exact features of viral particle propagation has a major impact in producing high virus yields in the development of vaccines. Use of lower MOI [0.01] could result in accurate, precise quantitative assays in virus diagnosis and titration methods