Construction of a novel recombinant vector carrying

زمینه و هدف: ‌لیشمانیوز به گروهی از بیماریهای انگلی ناشی از گونه های مختلف لیشمانیا اطلاق می شود که اشکال بالینی گوناگونی دارد. این انگل در انسان و حدود 100 گونه حیوان ایجاد بیماری می نماید و در بخش های وسیعی از جهان و از جمله ایران شایع است. یکی از زمینه های دستیابی به واکسنی مؤثر علیه لیشمانیا، دستکاری ژنتیکی این تک یاخته و استفاده از انگل مهندسی شده به عنوان واکسن می باشد. هدف از این تحقیق طراحی و ساخت کاست ژنی جدیدی است که به منظور وارد ساختن ژنهای خودکشی سلولی شامل HSV-tk و Yeast-cd به ژنوم لیشمانیا طراحی گردیده است. روش بررسی: در یک مطالعه آزمایشگاهی ابتدا دو قطعه ژن تیمیدین کیناز ویروس هرپس سیمپلکس (HSV-tk) و سیتوزین دآمیناز مخمر ساکارومایسس سرویزیه (Yeast-cd) به همراه ژن آلفا توبولین لیشمانیا (atub) با ترتیب tk-αtub-cd در پلاسمید pBluscript کلون شدند. سپس مجموعه ژنی مذکور از پلاسمید pBluscript خارج گردیده و در پلاسمید pF4X1.4sat کلون گردید و به منظور تأیید کانستراکت نهایی از هضم برش توسط آنزیم های محدود الاثر استفاده شد. یافته ها: قطعات ژنی tk-αtub-cd با موفقیت در پلاسمید pBluscript کلون گردیده و صحت کلونینگ مورد تأیید قرار گرفت. سپس این مجموعه ژنی در پلاسمید pF4X1.4sat درج شد و صحت حضور و ترتیب قرارگیری این ژنها اثبات گردید. نتیجه گیری: سیستم طراحی شده در این تحقیق می تواند دو ژن خودکشی سلولی را به درون ژنوم لیشمانیا منتقل کند و در تحقیقات آینده در زمینه دستیابی به واکسن زنده علیه لیشمانیا مورد استفاده قرار گیرد.

Expression of a Novel Chimeric Truncated t-PA in CHO Cells Based on in Silico Experiments

Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms

Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power

Abstract Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm

A dual drug sensitive L. major induces protection without lesion in C57BL/6 mice.

Leishmaniasis is a major health problem in some endemic areas and yet, no vaccine is available against any form of the disease. Historically, leishmanization (LZ) which is an inoculation of individual with live Leishmania, is the most effective control measure at least against cutaneous leishmaniasis (CL). Due to various reasons, LZ is not used today. Several live attenuated Leishmania have been developed but their use is limited. Previously, we developed a transgenic strain of L. major that harbors two suicide genes tk and cd genes (lmtkcd+/+) for use as a challenge strain in vaccine studies. These genes render the parasite susceptible to Ganciclovir (GCV) and 5-flurocytosine (5-FC). The dual drug sensitive strain of L. major was developed using gene targeting technology using a modified Herpes Simplex Virus thymidine kinase gene (hsv-tk) sensitive to Ganciclovir antibiotic and Saccharomyces cerevisae cytosine deaminase gene (cd sensitive to 5-flurocytosine) that were stably introduced into L. major chromosome. BALB/c mice inoculated with lmtkcd+/+ developed lesions which upon treatment with GCV and 5-FC completely healed. In the current study, the transgenic lmtkcd+/+strain was assessed as a live vaccine model to determine the time necessary to develop a protective immune response. C57BL/6 mice were inoculated with the transgenic lmtkcd+/+strain, and treated at the time of inoculation (day 0) or at day 8 after inoculation. Immunized animals were challenged with wild-type L. major, and complete protection was induced in mice that were treated at day 8. The results show that in contrast to leishmanization, in group of mice inoculated with a dual sensitive L. major development and persistence of lesion is not necessary to induce Th1 response and protection

C57BL/6 mice with history of <i>L. major</i> infection or group of mice which were inoculated with <i>lmtkcd^+/+ L. major</i> promastigotes and treated with GCV/5-Fcyt on day 0 or day 8 were subcutaneously (SC) challenged with 2×10⁶ wild type <i>L. major</i> along with a group of naive mice.

<p><b>A</b>. Lesion development was assessed by weekly measurement of footpad swelling. <b>B</b>. Parasite burden was quantified in spleen on week 5 post challenge. <b>C.</b> DTH reaction was checked by measurement of footpad swelling at 72 hours after injection of freeze-thawed <i>L. major</i> into the contralateral uninfected hind footpad. Five weeks after challenge, the splenocytes were cultured and stimulated <i>in vitro</i> with SLA (100 µg/ml), Con A (10 µg/ml), or with no stimulation for 72 hrs. <b>D & E</b> The supernatants were collected and the levels of IFN-γ (D) and IL-4 (E) were titrated using ELISA, <b>F.</b> Anti <i>Leishmania</i> IgG1 and IgG2a at 5 weeks post challenge. Presented data are representative of 2 independent experiments.</p

C57BL/6 mice were subcutaneously (SC) inoculated with either 2×10⁶ WT <i>L. major</i> or with <i>lmtkcd^+/+</i> parasites and were treated with GCV/5-FCyt on day 0 (<i>lmtkcd^+/+/D0</i>) or day 8 (<i>lmtkcd^+/+/D8</i>) or left untreated.

<p><b>A.</b> Lesion development was assessed by weekly measurement of footpad swelling. <b>B</b>. Parasite burden in groups of mice inoculated either with WT <i>L. major</i> or <i>lmtkcd^+/+</i> was assessed at week 10. <b>C</b>. Lymphocyte transformation test was done on spleen cells. <b>D</b>. production of IFN-γ using ELISA method. Presented data are representative of 2 independent experiments.</p

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios