Utilization of site-specific recombination in biopharmaceutical production

Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons

Frequency of VanA, VanB and VanH variants amongst vancomycinresistant enterococci isolated from patients in central region of Iran

Aim: The aim of this study was to investigate the VRE frequency and the rate of each gene in isolated enterococci frompatients with intestinal infection in the central region of Iran

Background: Enterococci infections are a public health growing concern due to the glycopeptide antibiotics resistanceespecially vancomycin. Genes, vanA, B, and H contribute to the influence of vancomycin-resistant enterococci [VRE]

Patients and methods: This study was conducted from January to July 2014 in Shahrood university hospital.Enterococci isolation and its antibacterial susceptibility were performed by culturing in Aesculin Azide agar and Kirby-Bauer method, respectively. Vancomycin-resistant genes were screened through conventional PCR, and subsequentlysequenced

Results: Among 265 specimens, 100 isolates revealed enterococci, in which E. faecalis [91%] and E. faecium [9%]. Theisolated enterococci were resistant to vancomycin [6%] and chloramphenicol [21%], whereas their large proportions [94% to100%] were multi-drug resistant. All VRE isolates belonged to E. faecalis, conversely, the E. faecium were susceptible to thesame antibiotic. Both vanA and vanH genes were identified in all VRE isolates, although, no vanB gene was indicated.Homology analysis of sequenced amplicons verified the full length compatibility to the worldwide reported genes

Conclusion: The present study revealed VR E. faecalis in gastroenteritis patients and resistance factor for vanA andvanH genes are coordinated. Since enterococci isolates were all multidrug resistance, increase in VR E. faecalis vanA /vanH in this area could be expected

[Evaluation of antibodies produced against lipopolysaccharides of vibrio cholerae for rapid detection of cholera]

Objective: Cholera is an endemic disease in Iran. Early detection, especially in times of disease outbreaks, is of vital importance. The antibody against the lipopolysaccharide [LPS] is an important method for bacterial detection. This study intends to extract and purify the LPS of Vibrio cholerae and evaluate the cholera antibody for detection purposes

Methods: Vibrio cholerae was cultured in tryptone extract medium. LPS was extracted by the hot phenol water method, purified, and dialyzed. We measured the LPS protein and sugar content, purity, and biological activity. Antibodies were produced by injection of the killed bacteria with Freund's complete adjuvant into rabbits and then the LPS was injected three times with Freund's incomplete adjuvant. After the last booster, blood samples were taken. We used ELISA to determine the antibody titers against the Inaba and Ogawa serotypes, the LPS of these serotypes, and several other similar bacteria

Results: The amount of protein in the purified LPS was approximately zero and sugar was 0.5 mg/ml. The LPS had a titer activity of 1024, and consisted of three bands [5.2, 4, and 5.14 KD]. Antibodies produced by the rabbits identified the bacterial Inaba and Ogawa serotypes, and the purified LPS. Ogawa and Inaba serotypes cross-react with each other but not with other species of Vibrio and other bacteria. The LPS antibody titer against the Ogawa serotype was 1:32000, whereas for Inaba it was 1:16000

Conclusion: Due to the low cost of production, high sensitivity, and importance of cholera diagnosis in Iran, the antiserum produced in this study can be used as a tool for early screening of cholera and discrimination of O1 strains from non-O1 strains in immunologic tests

Direct sequencing of cyclooxygenase-2 [COX-2] Revealed an Intronic Variant rs20 1231411 in Iranian patients with pancreatic cancer

There are hoarding documents for the biological importance of cyclooxygenase-2 [COX-2] in pancreatic carcinogenesis. We aimed to thoroughly investigate the DNA sequence variations of whole COX-2 exons in a large case- control study of pancreatic cancer by direct sequencing. The entire exonic regions of COX-2 including 10 exons were sequenced in the germline DNA of 96 patients with pancreatic cancer. Selected variants within exons six to seven [E6E7] amplicon from the test panel were genotyped in 96 controls. The COX-2 gene was demonstrated to be genetically conserved. Four missense mutations were found in three cases. However the common variant c.724-10_724-7deIATTT [rs20123141 1] that is located in intron 6, showed significant difference between cases and controls [21 [21.9%] vs 11 [%11.5], p=0.05]. This study determined that COX-2 has a conservative sequence, which is required for its enzymatic activity and supports the important role of this enzyme's expression in pancreatic cancer rather than any changes in its activity. The effect of intronic variant rs201231411 on COX-2 expression could be analyzed in future studies

[Sub-cloning of alpha subunit of anterior pituitary glycoprotein hormone into a mammalian expression vector]

Anterior pituitary glycoprotein hormones include thyroid stimulating hormone, lutinizing hormone, follicule stimulating hormone, and gonadotropine hormone. Each of them contains alpha and beta subunits. The alpha subunit gene is the same in all of these hormones and contains 4 exones and 3 intrones. The beta subunit is responsible for specific function of each hormone. The aim of this study was to clone alpha chain cDNA of glycoprotein hormones in a proper vector for eukaryotic system. To clone cDNA, alpha subunit of glycoprotein hormones was amplified by using one pair primers and T.vector as template and cloned in Not I and Bam HI sites of pcDNA3.1 plasmid. The recombinant plasmid transformed to E.coli Top10F? cell and colonies that contain plasmid were selected by Colony PCR. The accuracy of extracted plasmid of these clones was approved by enzyme digestion and sequencing. Enzyme analysis showed that pcDNA3.1-F351alpha had correct structure and sequencing confirmed by 100% homology of the gene with reported alpha gene in Gene Bank. Because of its proper structure, this plasmid is able to transform to Eukaryotic system and translation

Duane's retraction syndrome and karyotypic abnormalities / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:TO evaluate chromosome 8 abnormalities in Duane's retraction syndrome(DRS)type 1.METHODS:We evaluated chromosome 8 abnormalities in 29 consecutive cases of DRS type Ⅰ.DNA was isolated from the peripheral leukocytes of patients using a genomic DNA extraction kit,then D8S553 and D8S1797 markers used for polymerase chain reaction(PCR).RESULTS:None of the cases were positive for the two markers D8S553 and D8S1797 on chromosome 8 which were tested in our study.CONCLUSION:The possible cause of this finding is that DRS in our patients is more commonly sporadic rather than familial.We recommend study with more cases,other markers,and different chromosomes.