Expression, Solubilization, Refolding and Final Purification of Recombinant Proteins as Expressed in the form of "Classical Inclusion Bodies" in E. coli.

Escherichia coli has been most widely used for production of the recombinant proteins. Over-expression of the recombinant proteins is the mainspring of the inclusion bodies formation. The refolding of these proteins into bioactive forms is cumbersome and partly time-consuming. In the present study, we reviewed and discussed most issues regarding the recovery of "classical inclusion bodies" by focusing on our previous experiences. Performing proper methods of expression, solubilization, refolding and final purification of these proteins, would make it possible to recover higher amounts of proteins into the native form with appropriate conformation. Generally, providing mild conditions and proper refolding buffers, would lead to recover more than 40% of inclusion bodies into bioactive and native conformation.

Detection of West Nile virus by real-time PCR in crows in northern provinces of Iran.

BACKGROUND & OBJECTIVES: West Nile virus (WNV) is a positive-sense, single-stranded RNA virion, that belongs to the Flaviviridae family. This virus is preserved in a bird-mosquito cycle that is capable of inducing diseases as a dead-end or endpoint host in humans as well as horses. In 2016, a suspicious case of crow population death was reported by the Department of Environment, Ministry of Health, Iran. Considering the mass migration of birds together with the WNV-related symptoms, including uncoordinated walking, ataxia, inability to fly, lack of awareness, and abnormal body posture, it was necessary to further investigate the possible causes of this incident. The objective of this study was molecular detection of WNV in crows utilizing the real-time PCR method in the northern provinces of Iran. METHODS: A total of 12 crows (8 dead, 4 alive) with a possible WNV infection, were collected from the northern provinces of Iran (Golestan, Mazandaran, and Guilan). A tissue sample of the liver, kidney, or lung was collected from all the crows, and RNA was isolated using an RNA extraction kit. A one-step real-time PCR method using a TaqMan probe was used for virus detection. RESULTS: All the infected crows were positive for WNV. The 132-bp real-time PCR amplicon of the genome was detected in all the samples. Comparative phylogenetic analysis revealed that WNV isolated from Iran clustered with strains from the USA, Hungary, and Culex pipiens. INTERPRETATION & CONCLUSION: The WNV genome sequence was detected in all the infected crows. The results confirmed the connection of this isolation with clade1a strains. Hence, determining the epidemiologic and prevalence characteristics of the WNV for transmission control is of critical importance in Iran.

Novel, In-House, SYBR Green Based One-Step rRT-PCR: Rapid and Accurate Diagnosis of Crimean-Congo Hemorrhagic Fever Virus in Suspected Patients From Iran.

BACKGROUND: The Crimean-Congo hemorrhagic fever (CCHF) virus causes severe disease in humans, with a high mortality rate. Since, there is no approved vaccine or specific treatment for CCHF, an early and accurate diagnosis, as well as reliable surveillance, is essential for case management and patient improvement. OBJECTIVES: For this research, our aim was to evaluate the application of a novel SYBR Green based one-step real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay for the in-house diagnosis of the CCHF virus. PATIENTS AND METHODS: In this experimental study, the highly conserved S-region sequence of the CCHF viral genome was first adapted from GenBank, and the specific primers targeting this region were designed. Then, the viral RNA was extracted from 75 serum samples from different patients in eastern Iran. The sensitivity and specificity of the primers were also evaluated in positive serum samples previously confirmed to have the CCHF virus, by this one-step rRT-PCR assay, as well as a DNA sequencing analysis. RESULTS: From a total of 75 suspected serum samples, 42 were confirmed to be positive for CCHF virus, with no false-positives detected by the sequencing results. After 40 amplification cycles, the melting curve analysis revealed a mean melting temperature (Tm) of 86.5 ± 0.6°C (quite different from those of the primer-dimers), and the positive samples showed only a small variation in the parameters. In all of the positive samples, the predicted length of 420 bp was confirmed by electrophoresis. Moreover, the sensitivity test showed that this assay can detect less than 20 copies of viral RNA per reaction. CONCLUSIONS: This study showed that this novel one-step rRT-PCR assay is a rapid, reliable, repeatable, specific, sensitive, and simple tool for the detection of the CCHF virus.