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Investigation of Genetic Variations in Virulence Factor Genes ptxC, tcfA, and fhaB of Bordetella pertussis Clinical Isolates and Vaccine Strains in Iran

Volume 4, Issue 3 (Summer 2018)
Abstract
Aims: Pertussis is an important vaccine preventable disease. It is still a major cause of infant morbidity and mortality in the world. Although the incidence of pertussis was successfully reduced after vaccination, the resurgence of pertussis has been reported in many countries even with high vaccination coverage. Genetic variation in virulence factors is one of the important causes for pertussis reemergence. We investigated genetic characteristics and allele types of 3 important virulence associated genes, including ptxC, tcfA, and fhaB in clinical B. pertussis isolates collected from different provinces of Iran and vaccine strains.
Materials & Methods: Genomic DNA was extracted and ptxC, tcfA, and fhaB gene regions were amplified, using specific PCR primer. DNA sequencing was performed and data were analyzed.
Findings: ptxC2, tcfA2, and fhaB1 were the dominant alleles with 87.5%, 97.5%, and 97.5% frequencies, respectively. Vaccine strains B. pertussis 134 and B. pertussis 509 contain the genotypes ptxC2- tcfA2-fhaB1 and ptxC2- tcfA2-fhaB1.
Conclusion: Results for dominant alleles in ptxC2, tcfA2, and fhaB1 genes in Iran are consistent with dominant alleles of other countries such as Netherland, Finland, and Italy. It seems that ptxC2, tcfA2, and fhaB1 are the dominant circulating alleles in many countries after vaccination period, while vaccine strains have different alleles occasionally. More reported cases in recent years despite high coverage vaccination in Iran and genetic distances between clinical and vaccine strains suggest that antigenic changes in virulence factors possibly have an important role in the survival and evolution of the bacteria.

Recombinant expression of amyloid beta 1-42 (Aβ1-42) in Escherichia coli bacterial system
Pouran Badiri, Majid Sadeghizadeh, Bijan Bambai, S. Zahra Bathaie, Mehrnaz Bahrainy, Zarrin Minuchehr,
Volume 15, Issue 4 (10-2024)
Abstract

Introduction: Amyloid beta (Aβ) is the major constituent of harmful plaques in the Alzheimer’s patients. Thus, study of Aβ and understanding its related molecular and cellular mechanisms is essential for diagnosis and therapeutic interventions. This study introduces a rapid, simple, and cost-effective technique for production and purification of this peptide, utilizing the expression of Aβ gene within bacterial system.
Materials and methods: Aβ gene was synthesized and transferred into the expression vector pET26b. After induction by Lactose and  24 hours of incubation for Aβ expression the cell sediment was analyzed for presence of recombinant peptide using SDS-PAGE and Western blot.  Then the purification of recombinant peptide was carried using nickel chloride affinity chromatography. Characterization of purified Aβ was performed by evaluating cell cytotoxicity in 25 µM and 50 µM concentrations using MTT assay on Alzheimer cell line model SH-SY5Y.
Results: Colony PCR and sequencing results showed the correct insertion of Aβ coding fragment into the expression vector. Presence of bands with the expected size in the results of SDS PAGE and western blot had confirmed successful expression of his-tagged recombinant peptide. MTT assay results showed the purified peptide has respectively 30 and 50% cytotoxicity for 25 µM and 50 µM concentrations.
Discussion: Production of amyloid beta peptide in bacterial hosts seems to be favorable. Obtaining Aβ peptide in soluble phase is an important advantage of this study. Hence according to toxicity of the purified peptide, it can be utilized for cell line treatments and further researches on Alzheimer disease.
 


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