Restriction Endonucleases HindIII Research Articles

A Novel Technique for Constructing Infectious Cloning of Type 3 Porcine Circovirus.

Porcine circovirus type 3 (PCV3), which currently lacks effective preventive measures, has caused tremendous economic losses to the pig husbandry. Obtaining the strain of PCV3 is the key to preparing related vaccines and developing corresponding antiviral drugs. In this study, according to the linear sequence of PCV3 DNA published on GenBank, the sequence was rearranged with SnapGene gene-editing software, and after rearrangement, the HindIII restriction endonuclease site was added to the end of the linear DNA, so that both ends have the same restriction endonuclease site. On this basis, the rearranged linear DNA is obtained by gene synthesis, PCR amplification, DNA purification, etc., and is digested and connected in vitro to obtain cyclized DNA. PCV3 infectious clones were obtained by transfecting 3D4/21 cell lines. The obtained PCV3 was identified by PCR, Western blotting, and indirect immunofluorescence tests. The results showed that this study successfully obtained the strain of PCV3 in vitro. To further evaluate the pathogenicity of the obtained PCV3 infectious clones, this study established an animal model of Kunming mice infected with PCV3. The results of RT-PCR, Western blotting and immunohistochemistry showed that PCV3 can infect myocardium and alveoli of Kunming mice, but no PCV3 was detected in other tissues. The above studies indicate that PCV3 circular DNA can be used to construct PCV3 infectious clones. This research will provide a new method for the construction of circular DNA viruses and lay the foundation for the research and pathogenesis of PCV3 vaccine.

OSTEOCALCIN GENE POLYMORPHISM IN INDONESIAN MEN WITH PERIODONTITIS

Objective: Through this work, we aimed to clarify the association between the severity of periodontitis and osteocalcin (OC) gene C298T polymorphismin Indonesian male subjects.Methods: DNA was extracted from blood serum samples of 100 consenting Indonesian men whose periodontitis was classified as mild, moderate,or severe. Polymerase chain reaction and restriction fragment length polymorphism techniques were applied to evaluate the status of OC (C298T)polymorphism using HindIII restriction enzyme and electrophoresis in agarose gel to separate the indicated fragments.Results: Although genotype distribution of the OC C298T polymorphism showed a trend for more frequent occurrence of genotype hh (and allele h)with increasing severity of periodontitis, this trend was not statistically significant in the tested sample population, in whom the HH genotypeappeared to be relatively rare.Conclusion: The results showed no significant association between the severity of periodontitis and OC C298T polymorphism in Indonesian malesubjects.

ASSOCIATON OF KAPPA-CASEIN GENOTYPE AND THE LINEAR PARAMETER IN TWO INDIGENIOUS BOS INDICUS AND BOS TAURUS CATTLE IN NIGERIA

Kappa-casein as a mammalian milk protein is involved in a several important physiological processes and it’s about 80% of the total protein in cow milk. This study aimed at genotyping bovine Kappa casein (CSN3) in two indigenous Nigerian cattle populations and to determine the frequency distribution of Kappa casein variants as detected across the animals examined and their association with the body measurement. DNA was extracted from 100 blood samples of 50 White Fulani and 50 N’dama cattle for identification and genotyping of kappa-casein gene by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) test using HindIII restriction endonucleases. The PCR products of the specific primer K-F and K-R for the two cattle breeds give 530bp specific band. Digestion of 530bp amplified products of White Fulani and N’dama by restriction endonuclease HindIII generated three fragments of 530-, 370- and 160- bp each for the two breeds. Results of the cuts with this enzyme show the presence of genotypes AA, AB and BB in the samples. These findings suggest that BB genotype could be selected for increase body conformation and protein content of milk.
 Olanrewaju, B. M. | Department of Animal Production and Health, The Federal University of Technology Akure (FUTA), Ondo State, Nigeria.

Cloning,Sequencingand Analyzingof16S rRNA Gene from Inulin HydrolyzingBacteria

Gene of 16S rRNA is used for molecular identification of bacteria. This research aims to analyze the sequences of 16S rRNA gene from inulin hydrolyzing bacteria of UKG isolate. The 16S rRNA gene was isolated by PCR using BacF1 and UniB1 primers. The gene was cloned to pGEM-T Easy vector with E.coli TOP10F as host cell. Recombinant DNA was sequenced using T7 and SP6 primers. The size of the nucleotide base of the recombinant DNA of the sequencing result using SP6 primer was 1167 bp, whereas using T7 primer was 1218 bp. The both of the sequences overlapped 760 bp. The size of 16S rRNA gene in the sequences was found 1501 bp. Recombinant DNA that was restricted using EcoR1 showed 3 bands on agarose gel. They were 3000 bp, 800 bp, and 700 bp. The position of the EcoR1 palindrome sequence on the 16S rRNA gene was at the 832-837. The 16S rRNA gene also has been known by HindIII, BamHI, BalI, HaeIII, and SmaI restriction enzymes.

Genotyping of prolactin, kappa casein, and pituitary transcription factor 1 genes of the Anatolian water buffalo population in the Kızılırmak Delta

Thirty-five Anatolian water buffalo were selected from among a total of almost 7000 animals from 11 herds in the Kızılırmak Delta, which is known to be one of the most important areas for water buffalo farming in Turkey. The cleaved amplified polymorphic sequences (CAPS) method was used to identify single-nucleotide polymorphisms at 3 loci that are associated with milk production, namely PRL, CSN3, and PIT-1, which are associated with milk processing and milk yields, respectively. Sequence-tagged sites of PRL, CSN3, and PIT-1 were digested by restriction endonucleases RsaI, HindIII, and HinfI, respectively. Capillary electrophoretic analysis identified all samples as belonging to the GG and BB genotypes, which are undesirable genotypes for PRL and PIT-1 gene loci, but ideal for the CSN3 locus. Then the samples were sequenced by an ABI 3730 DNA Analyzer (Applied Biosystems). DNA sequencing of the amplified CSN3 fragment revealed one polymorphism at codon 135 (ThrACC > IleATC) in some samples. A homozygous structure was found for all three loci and the huge population loss between 1991 and 2008 suggests that a genetic bottleneck may have occurred in the population of Anatolian water buffalo in the Kızılırmak Delta.

Isolation and characterization of two novel plasmids pCYM01 and pCYM02 of Cylindrospermum stagnale.

Cyanobacteria play a vital role in supplying nitrogen into the soil and aquatic ecosystem. It has an extra chromosomal DNA, whose role is not yet defined well. Isolation and characterization of extra chromosomal DNA in cyanobacteria might help to understand its survival mechanism. Cylindrospermum stagnale isolated (and deposited in NRMCF 3001) from soil showed presence of four plasmids namely pCYLM01, pCYLM02, pCYLM03, and pCYLM04. The following plasmids pCYLM01 and pCYLM02 were subjected to restriction digestion using HindIII restriction enzyme and cloned into pBlueScriptSK(-) vector. The sequence of pCYLM01 contained 4 potential open reading frames (ORFs) that have amino acids in the range of 59–299. Among them, ORF1 shows high sequence homology to the bacterial replication initiator family protein as evident from BLASTP analysis. The analysis of 4359 bp plasmid pCYLM02 sequence revealed 7 ORFs which are longer than 50 amino acids in length. The ORF2 of pCYLM02 has 243 amino acids and is represented in the plasmid sequence from 3045 to 3776 bp. The ORF3 of pCYLM02 corresponds to the plasmid sequence from 2323 to 2976 and codes for a putative protein of 217 amino acids long. A number of small ORFs below 50 bp were also found in the sequence analysis.

Cloning and expression of the L1 immunogenic protein of human papillomavirus genotype 16 by using Lactobacillus expression system

Cervical cancer is the second most common cancer worldwide, after breast cancer. The agent of more than 90% of cervical cancer is Human papillomavirus (HPV). More than 100 various HPV with about 40 types of infective genotypes are identified and among these genotypes, near 15 carcinogenic genotypes are recognized as high risk. Moreover, genotypes 16 and 18 are the main agents of HPV related cancers. Today, HPV vaccines are available, but they could not cover all high-risk genotypes and also, they are really expensive. The aim of this study was to clone and express the L1 protein of human papillomavirus genotype 16 appropriately in the expression host Lactobacillus. The current study is the first step to prepare a live vaccine based on probiotic strain.In this study, the L1 gene of HPV genotype 16 was cloned to Lactobacillus pNZ7021 plasmid and its expression was assessed. So, the DNA of HPV genotype 16 was extracted from a clinical sample and by using specific primers, the L1 gene was amplified and inserted to the pBluescript cloning vector.Then, both amplified L1 gene and pNZ7021 were digested with BanII and HindIII restriction enzymes and ligated. Subsequently, the ligation product was transformed to Lactobacillus cremoris expression host. The recombinant host was cultured and L1 gene expression was evaluated by western blot method.The L1 gene amplifies by PCR successfully. The cloning of the L1 gene in the pBluescript vector was appropriate. The subcloning of the L1 fragment into the expression vector PNZ7021 was correct. Western blot results confirmed of L1 protein.It is suggested that the expression of the L1 protein-coding gene from human papillomavirus genotype 16 in the probiotic strain Lactobacillus cromoris could be considered as an immunogen, and this study needs further investigation.

Different vectors used to transform and clone of nonstructural NS1 gene of Influenza B in Escherichia coli

Flu is a highly contagious and common illness caused by influenza A, B, and C viruses. The aim of the present study was to investigate the transformation and cloning of NS1B gene with pET-32a, pET-32b and pQE-81L in Escherichia coli BL21(DE3) and DH5α. pUC57-NS1B synthetic gene was transform and clone in Escherichia coli BL21(DE3). Isolation, single digestion and ligation with pET-32b using HindIII restriction enzyme. Amplification of recombinant DNA was done with conventional PCR after transformation. Screening with IPTG of colonies. Gel electrophoresis was done for each step of cloning after isolation. Isolation, double digestion and ligation with pET-32a and pQE-81L using SacI, PstI and HindIII respectively.Recombinant DNA was attempted to be transformed into E. coli strains BL21 (DE3) and DH5α. pUC57 plasmid carrying NS1B gene was successful transformed and isolated from E. coli BL21 (DE3). Designed primers used for PCR of NS1B showed successful amplification. First screening of pET-32b-NS1B colonies using white/blue method, cloning NS1B into pET-32b using single restriction digestion with HindIII, pET-32a using double restriction digestion with SacI and HindIII and pQE-81L using double restriction digestion with PstI and HindIII gave unexpected result. This result may relate to re-ligation of digested vector for single digestion and uncompleted digestion for vectors of double restriction digestion. Current study has suggested that recombinant NS1B gene can be cloned using single digestion with other expression vectors.

A65 Characterization of endolysin gene of bacteriophages infecting Listeria spp. isolated from dairy industry wastewater

Bacteriophages and their endolysins, enzymes that degrade the cell walls of bacteria, are emerging as alternative tools to detect and inhibit growth of pathogen bacteria. Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a serious invasive disease that affects both humans and a wide range of animals. Listeria spp. are ubiquitous in the dairy farm environment and could be present in dairy-processing plants and wastewater. All Listeria-specific bacteriophages found to date are members of the Caudovirales, of the Siphoviridae or Myoviridae families. Myophages infecting Listeria have been recently classified by the ICTV in the Spounavirinae subfamily, as well as in the P100 virus genus. The aim of this work was to isolate Listeria spp. bacteriophages and their endolysin codifying genes from wastewater of a dairy industry. Wastewater with and without treatment was sampled during the course of a year, and isolation of bacteriophages was performed after an enrichment step using as hosts L. innocua, L. ivanovii, and L. monocytogenes serotypes 1/2a, 1/2b, and 4b. Bacteriophages infecting L. innocua and L. ivanovii were isolated (n = 24) from 3 out of 12 samples. Bacteriophages were purified, and the host range was determined using spot test and EOP against five collection strains and several field isolates of Listeria spp. Two bacteriophages of narrow and broad host range, vB_Lino_VEfB7, and vB_Liva_VAfA18, were selected for further characterization. High titer stocks of bacteriophages were purified by centrifugation with ammonium acetate, and morphological information on the purified bacteriophages was obtained by negative staining and transmission electronic microscopy. Their morphology, size, and contractile tails indicated that these bacteriophages belonged to the Myoviridae family. Bacteriophage genomes were extracted using phenol-chloroform, followed by ethanol precipitation, and tested by digestion with RNAsa A and DNAse I. RFLP was performed, digesting genomes with restriction enzymes HindIII and NcoI. Consistent with the morphological findings, bacteriophages contained dsDNA genomes but showed different RFLP patterns. A PCR designed to amplify conserved domains of endolysins—PGRP and CwlA—was applied to characterize this gene. Another PCR was designed to amplify the complete endolysin gene, and the complete sequence of this gene was obtained and analyzed. Substitution model selection and a maximum likelihood phylogenetic tree of the endolysin gene was carried out using IQ-Tree software. The sequences of the endolysin gene indicated that the codified enzyme is an N-acetyl-muramoyl-L-alanine amidase, related to A511 and P100 species of the recently described P100virus genus. Further evolutionary analyses are needed to evaluate their belonging to this species or their taxonomy within this genus.

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Synthesis a New Viral Base Vector Carrying Single Guide RNA (sgRNA) and Green Florescent Protein (GFP)

CRISPR/Cas9 system is a powerful gene editing tool in vivo and in vitro. Currently, CRISPR/Cas9 delivery cells or tissue with different vehicles are available, and Adeno- associated virus (AAV) in one of them. Due to AAV packaging size limitation, AAV base vectors that carry CRISPR/Cas9 system do not have florescent tag like GFP for simple detection and navigation of cells, containing AAV. The aim of this study was to modify and synthesis AAV base vector for CRISPR/cas9 system containing sgRNA and GFP.Px602 plasmid was double digested with NcoI and HindIII restriction enzyme. Gfp gene was amplified from px458 plasmid. Linear digested px602 and amplified Gfp gene were ligated together. After transformation and colony PCR on white colonies, plasmid was extracted and transfected to HEK-293 cell line. Gfp expression was monitored by florescent microscopy. After transfection of modified plasmid, florescent microscopy of HEK-293 cells showed shining green florescent cells, which indicate that Gfp gene, was replaced in the correct place according to our design.We modified an AAV base vector carrying CRISPR/Cas9 system, and synthesized a new vector carrying Gfp gene and sgRNA that can be packaged as reporter AAV for navigation and detection of cells, containing AAV.

Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy

R. Jimenez-Saiz, Y. Ellenbogen and K. Bruton et al. developed a method to identify bona fide IgE+ MBCs in humans, demonstrated their extreme rarity in circulation and cautioned against the clinical utility of their assessment.

A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase.

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

Genetic diversity of Ongole Grade, Aceh, and Sumbawa cattle based on polymorphism on ND-5 fragment mitochondrial DNA using PCR-RFLP technique

Abstract. Sutarno, Zahrah S, Astirin OP, Herawati E, Setyawan AD. 2019. Genetic diversity of Ongole Grade, Aceh, and Sumbawa cattle based on polymorphism on ND-5 fragment mitochondrial DNA using PCR-RFLP technique. Biodiversitas 20: 783-788. Genetic diversity is the basis of livestock breeding because it can be used as an initial improvement in livestock quality through artificial selection. This study aims to determine polymorphism in ND-5 fragment of mitochondrial DNA in Ongole Grade, Aceh, and Sumbawa cattle and their genetic diversity. The total DNA from the blood of the local cattle was extracted using the Wizard genomic DNA purification system from Promega and amplified using the PCR technique. The PCR product was then digested with HindIII enzyme using the RFLP technique to detect polymorphism. The genetic diversity of the Ongole Grade, Aceh, and Sumbawa cattle was analyzed using the formula from Nei and its genetic relationship was evaluated with the 2.02i NTSYSpc version program. Our findings showed there were polymorphisms in the ND-5 fragment of mitochondrial DNA. Digestion with HindIII restriction enzyme produces two types of haplotypes. Haplotype B is a 453 bp-sized DNA fragment that is not truncated by the HindIII enzyme, and haplotype A is a DNA fragment cut by HindIII enzyme into two with fragments of 336 bp and 117 bp. Polymorphism was found in Ongole Grade cattle, but not in Sumbawa and Aceh cattle. Haplotypes diversity in ND-5 fragments of mitochondrial DNA of Ongole Grade was 0.6250 while Sumbawa and Aceh cattle displayed no diversity of haplotypes. The genetic relationship shows that Sumbawa cattle belonged to the same cluster with Ongole Grade but separated from Aceh cattle.

Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method

BackgroundReliable measurement of DNA concentration and purity is important for almost all molecular genetics studies. Different plant species have varying levels of polysaccharides, polyphenols, and other secondary metabolites which combine with nucleic acids during DNA isolation and further affect the quality of the extracted DNA. The current extraction protocol is based upon the conventional cetyl trimethylammonium bromide (CTAB) method with further modifications for the extraction of DNA from variable plant seeds and crops belonging to seven different orders. The principle modifications currently employed for DNA extraction involved the use of higher CTAB concentration and higher levels of 2-β-mercaptoethanol. Additionally, higher concentrations of sodium chloride and potassium acetate were added simultaneously with absolute ice cold isopropanol for the precipitation of DNA free from polysaccharides.Results and conclusionThe prescribed modifications in the present method establish a quick and efficient standardized protocol for DNA extraction from different plant orders. The current extraction protocol, therefore, can be of great value for molecular analysis involving large numbers of different plant samples from different orders. These modifications consistently produced pure and high-quality DNA suitable for further molecular analysis. Successful PCR amplification with random amplified polymorphic DNA primer, NPTII gene, and the complete digestion of the isolated DNA with the HindIII restriction enzyme validated the quality of the isolated DNA. Moreover, it reflects the efficiency of the protocol and proves its suitability for further applications for the assessment of food safety, detection of genetically modified (GM) crops, and conservation of biodiversity.

Authentication of Sumateran Wild Boar (Sus scrofa vittatus) Meat Contamination by Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) Technique of Cytochrome b Gene

High population of Sumateran wild boar causes the increasing number of hunting and overflowing of wild boar meat availability in the market at a very low price. This condition leads to the falsification of meat. This study was aimed to characterize and differentiate Sumateran wild boar meat (Sus scrofa vittatus) and beef (Bos taurus) since Sumateran wild boar meat is often falsified as a beef in Indonesian traditional market. The domesticated pork meat (Sus scrofa domestica) was also investigated to compare Sumateran wild boar meat and domestic pork. Samples used in this study were DNA (deoxyribonucleic acid) from pork (Sus scrofa domestica), Sumateran wild boar meat (Sus scrofa vittatus), and beef (Bos taurus). Beef and pork were obtained from traditional markets in Bogor city whereas Sumateran wild boar meat was obtained from Way Kanan Regency, Lampung Province. Herein, we performed a thorough investigation on Sumateran wild boar meat (Sus scrofa vittatus) and beef (Bos taurus) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. This study utilized cytochrome b primer from mitochondria DNA with the size target amplification of 359 bp. This study utilized the restriction endonuclease enzymes, i.e. AluI, BsaJI, HindIII, RsaI, HaeIII, and TaqαI, in order to digest the amplification products from cytochrome b gene. The results showed that HaeIII is the merely specific enzyme to discriminate wild boar meat, domestic pork, and beef since HaeIII was able to digest these three meats in different locations of cyt b gene. However, AluI, BsaJI, HindIII, RsaI, and TaqαI enzymes were not specific enough to differentiate wild boar meat and domestic pork. In conclusion, among six tested enzymes, the use of PCR-RFLP analysis of the cyt b gene followed by digestion using HaeIII restriction enzyme provides a simple, relatively quick, and accurate identification of Sumateran wild boar species.

Identification of genetic marker for CSN3 gene in Karan Fries (Holstein Friesian crossbred) population

The study was conducted to identify genetic marker for CSN3 gene related to higher casein yield in 78 pedigreed Karan Fries (HF crossbred) cattle population at ICAR-NDRI, Karnal. PCR-RFLP of exon 4 region of CSN3 gene, carried out using HindIII restriction endonuclease, revealed three genotypes—CC, CA and AA with frequencies as 0.18, 0.67 and 0.15 and frequency of C and A allele as 0.51 and 0.49. Eight SNPs (C296A, C380T, C383T, G394T, C419A, G480A, A488T and G529T) were revealed through sequencing. Using Regression model, SNP (C419A) in the targeted region was found to contribute an increase of 1.8 g in κ-casein yield (CY) which was 2.81% of CY (63.62 g) in average test day milk yield (ATDMY). After screening progenies for SNPs based on Daughter-Dam Design, SNP G480A was found to be a potential genetic marker, contributing an increase of 7.5 g in CY (11.79% ofCY in ATDMY).

Production of MPT-64 recombinant protein from virulent strain of Mycobacterium bovis.

Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals which has been caused by a rod shaped, acid fast bacterium, called Mycobacterium bovis. The rapid and sensitive detection is a great challenge for TB diagnosis. The virulent strains of Mycobacterium tuberculosis complex (MTBC) have 16 different regions of difference (RD) in their genome which encode some important antigens. The major protein of M. bovis 64 (MPT-64) is one of the main immune-stimulating antigens which are encode by RD-2 region. The aim of the present study was cloning, expression and purification of MPT-64 as a protein antigen of M. bovis in a prokaryotic system for the usage in the future diagnostic studies. In this experimental study, the mpt-64 gene with 687 bp has been proliferated from M. bovis whole genome by polymerase chain reaction (PCR) method. The PCR product has been digested by BamHI and HindIII restriction enzymes and cloned into pQE-30 plasmid. The recombinant protein has been expressed in the Escherichia coli M15 with induction by isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed protein was analyzed on SDS-PAGE, and purified with Nitrilotriacetic acid (Ni-NTA) column. Finally, its biological properties were confirmed in Western blotting method using specific antibodies. Data showed the successful cloning of mpt-64 gene (as a 687 bp segment) in expression vector. The MPT-64 recombinant protein was ideally expressed and purified as a 24 kDa protein. The result of this study indicated that MPT-64 recombinant protein (24 kDa) has been successfully expressed and purified in a prokaryotic system, so this protein could be used for differential diagnosis of pathogenic and non-pathogenic Mycobacterium, in suspected BTB cases.

Molecular Characterization of Methicillin and Vancomycin Resistant Staphylococcus aureus Strains Isolated from Hospitalized Patients.

Objectives: Nosocomial infections caused by methicillin resistant Staphylococcus aureus (MRSA) and emergence of vancomycin resistant S. aureus (VRSA) have led to great concern in healthcare settings worldwide. Methods: A total of 100 S. aureus clinical isolates from hospitalized patients were investigated for antimicrobial susceptibility, the presence of resistance (mecA, vanA, and vanB) and virulence (hlaA, fnbpA, cna, clfA, tsst-1, eta, and spa) encoding genes, and molecular typing based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of spa gene. Results: All isolates were resistant to one or more antibiotics, with the most frequent resistance found against amoxicillin (69%). A total of 46 isolates were MRSA, and 40% of isolates were multidrug resistant (MDR). Of all S. aureus isolates, two isolates were confirmed as VRSA and four isolates confirmed as vancomycin intermediate S. aureus (VISA). The frequency of clfA, cna, tsst-1, and eta genes among MRSA isolates was significantly higher than methicillin sensitive S. aureus (MSSA). The significant correlation between MDR isolates and the carriage of multiple virulence genes was seen. All MDR isolates carried at least four virulence genes. Furthermore, biofilm formation in MRSA isolates was significantly higher than MSSA. The spa gene PCR products generated 4 major and 10 minor types. After digestion of spa amplicons with HindIII restriction enzyme, 10 different patterns ranging 174-938 bp were detected. S2b and S2a subtypes were detected frequently in MRSA isolates. Conclusions: It seems that the appropriate surveillance and control measures are essential to prevent the emergence and transmission of MRSA and VRSA strains in our country.

Probing the Function of Apolipoprotein A‐I Using Chimera Proteins

Human apolipoprotein A‐I (apoA‐I) is a 243 residue, 28 kDa protein that consists of an N‐terminal (NT) helix bundle domain and a less structured C‐terminal (CT) domain. ApoA‐I is the major protein component in high‐density lipoprotein (HDL) and is known to play an important role in reverse cholesterol transport. HDL promotes the removal of excess cholesterol from peripheral tissues to the liver. The CT domain of apoA‐I has a high affinity for phospholipids and initiates lipid binding, whereas the NT domain harbors the site of lecithin‐cholesterol acyltransferase activity. A recent study showed that removal of residues 231–243 led to a decrease in lipid binding affinity. Other studies have suggested that segments in the NT domain, residues 1–43 and 44–65, initiate lipid binding. Thus, conflicting data exists regarding the location of high‐affinity lipid binding in apoA‐I. A chimera protein was recently designed in which the CT domain of apoA‐I was attached to insect apolipophorin (apoLp‐III). The insect apolipoprotein acquired apoA‐I like properties, including high affinity lipid binding. Thus, this system allows for the identification of apoA‐I segments that have a high affinity for lipid surfaces. To identify potential lipid binding regions within apoA‐I, three chimeric constructs were designed using L. migratoria apoLp‐III. Residues 1–43 or 44–65 of apoA‐I were attached to the NT end of apoLp‐III. In addition, residues 231–243 of apoA‐I were attached to the CT end of apoLp‐III. The three DNA constructs were inserted into the pET‐20b (+) expression vector through restriction enzymes HindIII and NdeI. Site‐directed mutagenesis was conducted to introduce two cysteines into apoLp‐III at positions Thr‐20 and Ala‐149. The two cysteines lock the protein in an inactive state under oxidizing conditions to prevent lipid binding. This enables to study the lipid binding properties of the attached apoA‐I segments in the chimera proteins. Proteins are currently being expressed in Escherichia coli BL21(DE3) pLysS cells and will be purified through nickel‐affinity and size‐exclusion chromatography. The lipid binding properties will be assessed by the ability to solubilize phospholipid vesicles.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.