Cloning of c-Myc gene in embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent, self-renewing cells. These cells can be used in applications such as cell therapy, drug discovery, disease modelling, and the study of cellular differentiation. In this experimental study embryonic stem cells cultured in the laboratory and were amplified. Total RNA was extracted from cells and converted to cDNA by reverse transcription-polymerase chain reaction (RT-PCR). Then c-Myc gene was amplified by (PCR) and inserted into the pTZ57R/T vector. Ligated product was transformed into susceptible bacteria and transformed bacteria were screened on a selective medium. Plasmids extracted from bacteria and enzyme digestion to confirm the sequencing was performed. The cloned c-Myc gene can be used to prepare a gene cassette to produce stem cells from somatic cell.

A facile and cost-effective method for simultaneous preparation of lectin and tyrosinase from edible mushroom

<p><i>Agaricus bisporus</i> is a safe and rich source of tyrosinase and lectin. To address the increasing demand for these proteins, a facile method was developed for their simultaneous preparation from <i>A. bisporus</i>. The method includes demelanization of the raw extract followed by stepwise precipitation of the proteins from the aqueous extract. Employing the right organic solvent in demelanization and precipitation steps reduces the intermolecular interactions between these proteins. This enhances the efficacy of gel-permeation chromatography and results in preparation of partially purified lectin and tyrosinase suitable for many applications in biotechnology and bio-research. The method can be performed without diafiltration.</p

STUDY OF IMMUNOLOGICAL ACTIVITY AGAINST HAEMOPHILUS INFLUENZAE TYPE B PRP-DIPHTHERIA TOXOID CONJUGATE

ABSTRACT Haemophilus influenza is Gram-negative coccobacilli, Obligatory Anaerobic Bacteria without flagellum, without spores, positive catalase, and positive oxidase. Morphologically, polymorphism is well-known, especially in separated bacteria from old cultivars or GSF. They can be observed in different forms including coccoid, coccobacilli, bacilli, or filamentous forms. The main objective by the present study is to conjugate haemophilus influenza type b (Hib) polyribosylphosphate (PRP)-diphtheria toxoid through using different methods. Applied method at the present study would as follows: cultivation of heamophilus influenza in chocolate agar medium with factors X and -V; purification and precipitation of PRP from cultivation liquid through using alcohol precipitation; precipitation by N-Acetyl-N,N,N-trimethylammonium bromide GR (CTAB) Cetavlon; ultracentrifuge and purification by hydroxyapatite; filtering through 22% m and distribution in sterile vial and lyophilizing it; producing diphtheria toxoid from Razi Factory and dialyzing it with dialyze packet of cutoff 10000 in order to pull out preservative and filtering it to 22% m and distributing it in sterile vial and then lyophilizing it; conjugation by diphteria toxoidusing ADH and cyanogen bromide as the separating factor and EDAC as the conjugation factor; measuring amount of protein through Lory and Bradford Tests; electrophoresis; measuring amount of polysaccharide through Ribose Test and quellung test; identification of fraction of conjugation and calculation of conjugation output; injection of conjugation and PRP just to several white laboratory rabbits for 2 times with interval of 15 days with aluminum hydroxide adjuvant; taking blood samples and finally investigation of immunology of collected bloods from injection of conjugation and PRP using serum bactericidal test. At the present study, serum titers in serum bactericidal test would be respectively equal to 1.8, 1.16, and 1.32 for the first group;1.4, 1.8, and 1.16 for the second group; and finally 1.16, 1.32, 1.64 for the third group. PTHOGENICITY Strains without Haemophilus influenza capsule would usually cause infection in previously damaged tissues;for example, inpatients with chronic bronchitis or cystic fibrosis. Cell CF has a key role in connecting these strains to nasopharyngeal mucosa cells. External membrane proteins are also effective in connection and colonization of the bacteria on the mucosa surfaces. Usually, primary focus of the infection, resulted from strains non-capsular strains, would be appeared in nose and throat; although the infection may be expanded to other parts like sinuses or Eustachian tube in ears. Noncapsular strainswould cause usually diseases such as sinusitis, pneumonia, otitis,mastoiditis, conjunctivitis, endometritis, pericarditis, urinary tract infection (UTI), etc. In capsular strains, especially type b, the main role of the capsule is preventing phagocytosis of Poly Morpho Nuclears and also serum resistance of bacteria. Capsular strains would mainly cause invasive infections in human body such as meningitis, epiglottitis, pneumonia, cellulite, laryngitis, osteomyelitis, septic arthritis, and septicemia. Primary focus of infection in capsular strains is usually in nasopharynx. The bacteria would be connected to nasopharyngeal mucosa cells by external membrane proteins. The bacteria can enter to blood flow and then to different organs of infants, individuals with immunity disorder, cancer patients, organ recipients, and other similar individuals. The most important infectious disease that may be resulted from such strains is meningitis, in which LOS pieces and bacterial peptidoglycan can lead to Presence of antibody against exterrnal membrane proteins and capsule plays a key role in preventing localized and disseminated infections. Protective antibody against capsule can be transferred to the infant throughout the placenta and can protect the infant for 6 months after birth day against invasive infections, resulted from Haemophilus influenza. After the mentioned age, protective antibody would be significantly decreased and then infant would become vulnerable against disseminated and invasive infections, especially meningitis. Hence, vaccination is so significant in age range from 2 to 12 months old. In immunology of infectious diseases, in order to activate T cell, assistant t cell co-receptor (CD4) would identify supplied specific peptide beside the MHC class II and then would transfer identification signal to native T cell. It should be mentioned that 2 signals are requiredin order to activate T cell. The first signal is same mentioned identification signal. Second signal would be presented through interleukin -2 (IL -2). Through identifying the antigen, T cell would be activated and then would enter to cell cycle. At this stage, IL-2 and receptor can construct it; although in order to continue its activity and increase its clones, the two mentioned components should be connected to each other. In T cell (NFAT), action of IL-2 should be copied, which would be possible through connecting CD28-B7 that both of them are from immunoglubulin super family and are known as Costimulation. Clones of T cell would be increasethrough transferring activity signal of a series of biochemical events inside T cell such as increase in amount of ionized calcium, increase in protein production, and increase in clones. Assistant T cells would identify complex peptide and MHC on surface of B cell and would activate it against polysaccharide in order to produce antibody. EPIDEMIOLOGY Haemophilus influenza is a pathogenic microbe, especially in age range of 3-18 months. At these ages, immune system would not act desirably against independent antigens from thymus like haemophilus influenza type b polysaccharide. In order to make effective vaccine and consuming it by infants, its capsul antiogen would be connected to a protein transporter in conjugated form and then produced vaccine would be applied. Immune function of the mentioned vaccinehas been reported to 80% because of imposing effect through T cells against invasive form of Haemophilus influenza. Hence, its application is significant in routine vaccination of infants. Bacterial meningitis is an important problem in kids and infants. Over the decade, significant advances have been achieved for rapid diagnosis of the meningitidis disease such as serologic methods and antigen seeking methods. Etiologic factors of bacterial meningitis are relatively various and most scholars have introduced several factors as the main factors of bacterial meningitis, especially in childhood, including Haemophilus influenza, Neisseriameningitidis, andStreptococcus pneumonia. According to relevant studies, the mentioned factors can be varied based on time, location, and age of patients. Vanger et al (1990) have conducted a study in America and have found that the most common separated bacteria of infants are respectively Streptococcus pneumonia to 40%; Haemophilus influenza type b to 18%; and Neisseriameningitidis to 14%. However, in another study by Ronald Gold et al (1992) Bacterial suspension was transferred to 2L containers and was then centrifuged for 1h with 3500rpm. Supernatants were collected and precipitated cells were disposed. Collected supernatants were maintained in the refrigerator for on night under temperature of 4ºС. About 9L of supernatants were distributed in 4 alonjes, 3 alonj containing 2.5L supernatant and one alonj containing 1.5L supernatant. Per every 2.5L supernatant, about 6L and 750cc ethanol 96% was added. Then the supernatant was maintained for one night under temperature of 4 ºС. During the maintenance time, the PH was regulated on 7.3 and glacial acetic acid was added to it and then pH ratio achieved 6.8. Then, it was maintained again for 24h under temperature of 4ºС, so that polysaccharides could be precipitated. Washingbychloro-magnesium Contents of alonjes were centrifuged for 1h with 3000rpm. Supernatants were disposed and obtained precipitation was maintained. Then, 1.8L chloromagnesium with density of 0.05 was added to it and was mixed properly, so that sediments could be solved completely. It was then centrifuged for 1h with speed of 3000rpm and sediments were disposed and supernatant was maintained