همسانه‏سازي و تعيين توالي ژن گليكوپروتئين G ويروس تب بي‏دوام گاوي در اشرشياكلي

Cloning and sequencing of G glycoprotein gene of bovine ephemeral fever virus in Escherichia coli

تب بی‏دوام گاوی (BEF) یك بیماری ویروسی قابل‌انتقال توسط بندپایان در گاو و گاومیش می‏باشد كه در مناطق گرمسیری و نیمه‌گرمسیری آسیا، استرالیا و افریقا پراكنده شده است. در سال‏های اخیر این بیماری در بسیاری از استان‏های ایران نیز شایع شده و موجب بروز خسارت‏های اقتصادی گردیده است. در این مطالعه ژن گلیكوپروتئین G ویروس تب بی‏دوام گاوی پس از تكثیر با واكنش PCR در ناقل pTZ57R/T كلون و تعیین توالی شد. پس از اطمینان از صحت همسانه‏سازی، محصول PCR با استفاده از آغازگر‏های دارای جایگاه برشی آنزیم BamHI تكثیر و به پلاسمید بیانی pMalc2x انتقال داده شد و سپس به سلول‏های پذیرا شده اشرشیاكلی (سویه‏های ‏DH5α و Rosetta) منتقل گردید. غربال‌گری كلونی‏های باكتریایی حاوی پلاسمید نوتركیب با روش PCR و هضم آنزیمی، نشان از ورود موفقیت‏آمیز سازه pMalc2x-G به اشرشیاكلی داشت. این مطالعه اولین گزارش از همسانه‏سازی ژن گلیكوپروتئین G ویروس تب بی‏دوام گاوی در ایران است كه می‏تواند پایه‏ای برای تولید واكسن و ساخت كیت تشخیصی الایزا برای این بیماری در مطالعات آینده باشد.

Introduction Bovine ephemeral fever (BEF) is an arthropod-borne viral disease of cattle and water buffalo, spanning tropical and subtropical zones in Asia, Australia, and Africa continents. The clinical signs of BEF disease in cattle are acute febrile reaction, stiffness, lameness, depression, cessation of rumination, and constipation. This disease was caused by Ephemerovirus of the Rhabdoviridae. Bovine ephemeral fever virus (BEFV) has a negative single stranded RNA genome and viral particles are bullet or cone-shaped. Five structural proteins of BEFV have been described comprising a nucleoprotein (N), a polymerase-associated protein (P), a matrix protein (M), a large RNA-dependent RNA polymerase (L) and a surface glycoprotein (G) spanning the viral envelope. The G protein is a class I transmembrane glycoprotein that forms clear projections on the virion surface. The protein G is main protective antigen and there are four antigenic sites (G1, G2, G3, and G4) on its surface. The G glycoprotein of BEFV is a type-specific neutralizing antigen and induces protective immunity in cattle. It has been shown to induce virus-specific neutralizing antibodies that confer passive protection against intracerebral infection of suckling mice and protect cattle against experimental intravenous BEFV challenge. In recent years, BEF has been distributed in many provinces of Iran such as Tehran, North Khorasan, Golestan, Mazandaran, Ardebil, Ilam, Khuzestan, Fars, and Yazd and caused economic losses. Treatment will be very effective if BEF is diagnosed early. The blocking ELISA test is preferred for the diagnosis and monitoring of clinical bovine ephemeral fever. On the other hand, vaccination has been suggested as an effective approach for control and prevention against to BEF. Nevertheless, no effort has yet been accomplished for production of a vaccine and development of an ELISA kit for BEF diagnosis in Iran. Hence, the aim of this study was molecular cloning and sequencing of G glycoprotein gene of bovine ephemeral fever virus (BEFV) in Escherichia coli. These findings provide the basis for the production of a vaccine and development of an ELISA kit for BEF diagnosis in future studies. Materials and method The strain of BEFV used in this study was provided of Razi Vaccine and Serum Research Institute (Hesarak, Karaj, Iran). BLAST analysis based on G gene sequence showed that this strain had the most identity with the YHL strain isolated in Japan’s Yamaguchi prefecture in 1966. DH5α and Rosetta Strains of E. coli were used for cloning of G gene. Total RNA was extracted from the supernatant of BEFV-infected cells and reverse-transcribed into cDNA. The cDNA template was amplified by PCR using the primer pairs GF and GR in order to amplify the full length of G gene. The amplified G fragment was cloned into pTZ57R/T vector and then transferred into E. coli DH5α. Insert-positive clones were isolated by blue/white screening of bacterial colonies using X-gal and IPTG on LB ampicillin plates. The transformed colonies were screened for desired gene performing colony PCR. In order to confirm of cloning accuracy, the recombinant plasmids were sequenced. Then a recombinant pTZ57R/T plasmid (pTZ57R/T-G) was used as the template for PCR amplification using primers containing the BamHI restriction endonuclease sequence. A pMalc2x vector was used in this study. In order to introduce the amplified gene into pMalc2x vector, plasmid DNA and purified PCR products were digested with BamHI restriction enzyme through the sites created by the primers. After ligation, pMalc2x-G construct was transformed into susceptible E.coli (DH5α and Rosetta strains) cells. Results and Discussion The 1823 bp fragment of G gene was successfully amplified in RT-PCR and visualized on 1.5% agarose gel. The transformed DH5α colonies (with recombinant pTZ57R/T vector) were confirmed and selected for having 1823 bp fragment using colony PCR method. The result of sequencing of pTZ57R/T-G showed that the predicted amino acid sequence of G gene had four amino acid substitutions in sequence that have no significant effect on antigenic sites. Amplification of a recombinant pTZ57R/T plasmid (pTZ57R/T-G) using primers containing the BamHI restriction endonuclease sequence produced an amplified fragment of G gene to introduce into pMalc2x vector. After verification, the recombinant plasmid was extracted and then transformed into E. coli Rosetta competent cells. Screening of bacterial colonies containing recombinant plasmid by PCR and restriction enzyme digestion showed that pMalc2x-G construct was successfully transformed into E.coli. Conclusion This study is the first report of cloning of G glycoprotein gene of bovine ephemeral fever virus using pTZ57R/T and pMalc2x vectors into E.coli in Iran. The pMalc2x-G construct can be used for production of G recombinant protein for development of an ELISA kit for diagnosing BEF in future studies. Furthermore, each of these recombinant plasmids can be used as the basis for transformation and expression of G gene into a eukaryotic expression system in order to production of a vaccine for BEF in future studies.