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Bemisia tabaci is a species complex that causes damage to its broad range of plant hosts through serious feeding. It transmits plant viruses of different groups to important agricultural crops. Some important cash crops of Pakistan are sugar cane, rice, tobacco and seed oil. It shows high genetic variability and is differentiated as races or biotypes. Biotypes are, biotype Q, biotype B, biotype B2, biotype M, biotype L, biotype A, biotype H, biotype C, biotype K, biotype N, biotype R, biotype E, biotype P, biotype J, biotype S, biotype AN. Although the current report based on the Bayesian study of mitochondrial cytohrome oxidase gene1 (CO1) DNA sequences has classified the different populations of whiteflies into twelve genetic groups which are Mediterranean, Sub-Saharan Africa silverleafing, Indian Ocean, Asia II, Asia I, Australia, New World, Italy, China, Sub-Saharan Africa non-silverleafing, Mediterranean/Asia Minor/Africa and Uganda sweet potato. Begomoviruses is largest group of viruses transmitted by B. tabaci and cause major diseases of crops such as tomato and chili leaf curl disease, cassava mosaic disease; yellow mosaic disease of legumes and cotton leaf curl disease. The main objective of current study is to inculpate knowledge regarding genetic diversity of whitefly in cotton fields across Pakistan via analysis of partial DNA sequence of mitochondrial gene Cytochrom Oxidase I (mtCO1).

Studying the developmental genetics of plant organs requires following gene expression in specific tissues. To facilitate this, we have developed dual expression anatomy lines, which incorporate a red plasma membrane marker alongside a fluorescent reporter for a gene of interest in the same vector. Here, we adapted the GreenGate cloning vectors to create two destination vectors showing strong marking of cell membranes in either the whole root or specifically in the lateral roots. This system can also be used in both embryos and whole seedlings. As proof of concept, we follow both gene expression and anatomy in Arabidopsis (Arabidopsis thaliana) during lateral root organogenesis for a period of over 24 h. Coupled with the development of a flow cell and perfusion system, we follow changes in activity of the DII auxin sensor following application of auxin., A vector system and flow cell set-up allow long-term imaging of both gene expression and anatomy in Arabidopsis primary and lateral roots.

Clostridium difficile is a spore-forming gram-positive bacterium, recognized as the primary cause of antibiotic-associated nosocomial diarrhoea. Clostridium difficile infection (CDI) has emerged as a major health-associated infection with increased incidence and hospitalization over the years with high mortality rates. Contamination and infection occur after ingestion of vegetative spores, which germinate in the gastro-intestinal tract. The surface layer protein and flagellar proteins are responsible for the bacterial colonization while the spore coat protein, is associated with spore colonization. Both these factors are the main concern of the recurrence of CDI in hospitalized patients. In this study, the CotE, SlpA and FliC proteins are chosen to form a multivalent, multi-epitopic, chimeric vaccine candidate using the immunoinformatics approach. The overall reliability of the candidate vaccine was validated in silico and the molecular dynamics simulation verified the stability of the vaccine designed. Docking studies showed stable vaccine interactions with Toll‐Like Receptors of innate immune cells and MHC receptors. In silico codon optimization of the vaccine and its insertion in the cloning vector indicates a competent expression of the modelled vaccine in E. coli expression system. An in silico immune simulation system evaluated the effectiveness of the candidate vaccine to trigger a protective immune response.

Starch is a natural stored carbohydrate resource for the human diet, synthesized and stored in amyloplasts in higher plants. Starch synthase III is directly linked to the amylopectin chain’s elongation in wheat grains, but global warming is a critical barrier to its activity. The present investigation includes the characterization of the TaSS-IIIa1D gene in two wheat genotypes, viz. PBW-343 (heat susceptible) and IC252874 (heat tolerant). The genomic DNA of the above genotypes was used for PCR amplification of TaSS-IIIa1D gene using overlapping gene-specific primers and cloning the gene using the pJET1.2/blunt cloning vector, followed by sequencing and detection of SNPs. In-silico analysis revealed that the TaSS-III gene contains two homologs, TaSS-IIIa and TaSS-IIIb, which were found on the plus strand of chromosome 1 (1A, 1B and 1D) and minus strand of chromosome 2 (2A, 2B and 2D), respectively. The genomic size of the TaSS-IIIa1D gene was approximately 10,529 bp. All homeologous copies of the gene contained 16 exons. Twenty-nine specific SNPs were identified in PBW343 and twenty in IC252874 genotypes for the gene vis-a-vis with the reference copy of Chinese spring cultivar. There were 14 intronic and 15 exonic SNPs contributing to 18 transitions and 9 transversions in the genotype PWB-343, reflecting the transition bias, while in genotype IC252874, nine transitions, nine transversions, and two deletions contribute to 6 intronic and 14 exonic SNPs showing no such bias. Eighteen SNPs detected between the two genotypes for the gene consisting of 11 transitions and 7 transversions also showed transition bias. Third exon found most variable with the maximum number of SNPs. Seven SNPs were found associated with SBD-1, SBD-2, and SS-CD domains of the TaSS-IIIa1D protein. SNPs identified between the heat-sensitive and heat-tolerant genotypes for the heat-sensitive enzyme coding gene could be utilized for wheat improvement after proper validation.

The four hydrogenase isozymes; hydrogenase 1 (Hyd-1), hydrogenase 2 (Hyd-2), hydrogenase 3 (Hyd-3) and hydrogenase 4 (Hyd-4) of Escherichia coli have been reported for their crucial functions in the hydrogen metabolism; however, their distinctive roles could not be completely understood. In this study, four ideal hydrogenase operon mutants, Δhyb hyc hyf, Δhya hyc hyf, Δhya hyb hyf, and Δhya hyb hyc, in which only a single hydrogenase is intact in the genome, were constructed as well as one quadruple mutant (Δhya hyb hyc hyf) that all four hydrogenase operons were deleted. First, single operon mutants and single-gene mutants for each hydrogenase showed different hydrogen productivity and growth in the anaerobic fermentation, indicating that bacterial phenotype regarding the hydrogen metabolism via the deletion of each operon is different with that of each single gene. Then, 4 triple hydrogenase operon mutants and one quadruple mutant were investigated to evaluate the hydrogen metabolism (hydrogen production and uptake) using glucose or glycerol as a substrate of hydrogen fermentation. With both the carbon sources, only Hyd-2 and Hyd-3 were able to produce hydrogen. Furthermore, all the hydrogenases showed hydrogen uptake activity. In addition, no hydrogen production and hydrogen uptake were detected in the quadruple mutant which does not have all 4 hydrogenases. Hydrogen production from Hyd-2 and Hyd-3 was further confirmed by complementing their operons in the cloning vector pBR322.

Aim: The objective of this study was to produce recombinant protein GRA-4 (rGRA-4) of a local Toxoplasma gondii isolate as a candidate for a toxoplasmosis diagnosis kit in Escherichia coli BL21 (DE3) competent cells using pET SUMO plasmid. Materials and Methods: Samples used were stock T. gondii tachyzoites DNA from the Parasitology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta. Amplified GRA-4 polymerase chain reaction product of T. gondii tachyzoite DNA was cloned in the pET-SUMO TAR cloning vector. The GRA-4 gene from T. gondii local isolate was sequenced, followed by plasmid transformation, recombinant plasmid DNA isolation, and recombinant protein expression in DE3 competent cells. Results: The amplification product of GRA-4 T. gondii gene was 1036 bp, with 48 kDa molecular weight after expression in DE3 competent cells. An alignment of the amino acid sequence of GRA-4 from the local isolate which was cloned with GRA-4 was obtained from NCBI database and showed 99.61% homology to the predicted GRA-4 from the T. gondii Izatnagar isolate. Amino acid sequence of the predicted GRA-4 protein from local isolate was different at positions 19 and 304. Conclusion: This research cloned rGRA-4 in pET SUMO plasmid.

Here we report a simple and flexible method for DNA data storage based on Perl script. For this approach, the text data of the preamble of the “Universal Declaration of Human Rights” consisting of 2,046 words was encoded into the corresponding 8,148 base pairs of DNA using Perl-based encoding with a hash table. The encoded DNA sequences were then artificially synthesized for storage. The information DNA consisted of a total of 22 chemically synthesized DNA fragments with 400 nucleotides each, which were inserted into a cloning vector to multiply the plasmid DNA. The nucleotide integrity of the data-carrying DNA sequences were ensured under the accelerated aging conditions. Also, an erroneous nucleotide in the information DNA sequences was successfully corrected using the overlap extension PCR method. The stored DNA was read by sequencing, and the resulting DNA sequence information was successfully decoded to convert the DNA records back to the original document. Our results indicate that textual data can be stored in DNA using a simple, easy, and flexible Perl by running a script from the command line.

Alteration in two-component systems (TCSs), which are signal transduction pathways in prokaryotes, can result in antibiotic resistance. Recently, it has been shown that the overexpression, using a multicopy cloning vector, of the dcuR, rcsB, and yehT genes, which code for the response regulator (RR) part of TCSs, enhanced the minimal inhibitory concentrations (MICs) of carbapenems in Escherichia coli K-12 derivative KAM3. Herein, the contribution to carbapenem resistance of the DcuS/DcuR, RcsC/RcsB, and YehU/YehT TCSs was assessed in E. coli K-12 derivative BW25113 (A phylogroup) and 536 (B2 phylogroup) recipient strains in combination with extended-spectrum β-lactamase that exhibit a weak carbapenemase activity. The genes encoding both the sensor kinase (SK) and the RR, on the one hand, and the genes encoding the SK only, on the other hand, of these regulating pathways were disrupted. Subsequently, the mutants and their parental strains were transformed by a recombinant plasmid encoding the CTX-M-15 gene, before testing their susceptibility to carbapenems and their fitness. Results showed a trade-off between enhanced MICs for ertapenem, which remained above the clinical resistance breakpoint, and decreased growth rate, specifically for the 536 strain SK mutants. In conclusion, mutations in dcuS/dcuR, rcsC/rcsB, and yehU/yehT genes may be a pivotal first-step event in the development of carbapenem resistance.

Σκοπός της διατριβής ήταν η κλωνοποίηση της κωδικής αλληλουχίας του γονιδίου της ποντικίσιας β-σφαιρίνης. Εξετάσαμε το μοριακό υπόβαθρο της κληρονομικής ασθένειας της Β-θαλασσαιμίας, καθώς και τις υπάρχουσες θεραπευτικές προσεγγίσεις. Στη συνέχεια, έγινε κλωνοποίηση της κωδικής αλληλουχίας του γονιδίου της ποντικίσιας β-σφαιρίνης σε δύο διαφορετικούς φορείς κλωνοποίησης. Ακόμη, έγινε παραγωγή της ανασυνδυασμένης πρωτεΐνης από βακτηριακό σύστημα έκφρασης. Ένα άλλο θέμα που θίξαμε ήταν η διαλυτοποίηση των αδιάλυτων βακτηριακών εγκλείστων που περιείχαν το πρωτεϊνικό εκχύλισμα. Ασχοληθήκαμε επιπλέον με την ηλεκτροφόρηση και την ανοσοαποτύπωση της πρωτεΐνης-ενδιαφέροντος σε πηκτή πολυακρυλαμιδίου και ειδική μεμβράνη αντίστοιχα, όπου εντοπίστηκαν υδρολυμένα τμήματα της μονομερούς και της διμερούς μορφής της πρωτεΐνης., The aim of the present thesis was the cloning of CDS of murine β-globin for production of the recombinant murine β-globin protein. We examined the proposed therapeutic approaches and the molecular basis of Β-thalassemia. Then we proceeded to the cloning of CDS of murine β-globin in two different cloning vectors. Furthermore, we produced the recombinant murine β-globin protein from a bacterial expression system. We also examined the solubility of the inclusion bodies, that contained the produced protein, in L-arginine. Another aim of this study was the electrophoresis and immunoblotting of the produced recombinant protein of interest in polyacrylamide gel and polyvinylidene fluoride membrane, respectively.

The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials.HighlightspOPIN-GG expression vectors allow for modular cloning enabling rapid screening of purification and solubility tags at no loss of expression compared to previous vectors.Cloning into the pOPIN-GG vectors can be performed from PCR products or from level 0 vectors containing the required parts.Several vectors with different resistances and origins of replication have been generated allowing the effective co-expression and purification of protein complexes.All pOPIN-GG vectors generated here are available on Addgene, as well as level 0 acceptors and tags.

Aspergillus oryzae is a safe microorganism that is commonly used in food production. We constructed a self-cloning vector capable of high expression in A. oryzae. Using the vector, three putative pectin methylesterase (PME) genes belonging to Carbohydrate Esterase family 8 derived from A. oryzae were expressed, and several characteristics of the gene products were examined. The effects of temperature and pH on the three enzymes (AoPME1, 2, and 3) were similar, with optimal reaction temperatures of 50 - 60 °C and optimal reaction pH range of 5 - 6. The specific activities of AoPME1, 2, and 3 for apple pectin were significantly different (34, 7,601, and 2 U/mg, respectively). When the substrate specificity was examined, AoPME1 showed high activity towards pectin derived from soybean and pea. Although AoPME2 showed little activity towards these pectins, it showed very high activity towards apple- and citrus-derived pectins. AoPME3 showed low specific activity towards all substrates tested. Sugar composition analysis revealed that apple- and citrus-derived pectins were rich in homogalacturonan, while soybean- and pea-derived pectins were rich in xylogalacturonan. When pea pectin was treated with endo-polygalacturonase or endo-xylogalacturonase in the presence of each PME, specific synergistic actions were observed (endo-polygalacturonase with AoPME1 or AoPME2 and endo-xylogalacturonase with AoPME1 or AoPME3). Thus, AoPME1 and AoPME3 hydrolyzed the methoxy group in xylogalacturonan. This is the first report of this activity in microbial enzymes. Our findings on the substrate specificity of PMEs should lead to the determination of the distribution of methoxy groups in pectin and the development of new applications in the field of food manufacturing.

This study was conducted to determine the heterologous expression of eukaryotic gene in Escherichia coli variable. The expression of Pakistani buffalo (Bubalus bubalis) and cow (Bos taurus) proinsulin genes in BL21 codon plus cells is 30% of total cellular protein. Total RNA was isolated from pancreatic tissues of local (Pakistani) breeds of buffalo (B. bubalis) and cow (B. taurus), converted to cDNA and cloned in a T/A cloning vector. There are five silent mutations: one in B-chain, two each in A-chain and C-peptide encoding regions of local Pakistani buffalo proinsulin DNA sequence as compared to the internationally reported cow proinsulin sequence. The DNA sequence of local (Pakistani) cow proinsulin shows there is one nucleotide encodes C-peptide that has mismatch with the reported cow proinsulin sequence but mismatched nucleotide is same between Pakistani cow and Pakistani buffalo proinsulin sequence. This indicates the genetic similarity of Pakistani cow with Pakistani buffalo. Both genes were expressed in BL21 Codon plus (DE3)-RIL cells with 0.2 mM IPTG at 37°C for 6 to 8 h. Proinsulin was expressed as 30% of total cellular proteins as insoluble inclusion bodies. Key words: Bubalus bubalis, Bos taurus, cDNA, T/A cloning vector, BL21 Codon plus (DE3)-RIL cells.

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins. Within this host, the choice of cloning vector constitutes a key factor for a satisfactory amplified expression of a target gene. We aimed to develop novel, unpatented expression vectors that enable the stable maintenance and efficient overproduction of proteins in E. coli. A series of expression vectors based on the ColE1-like pIGDM1 plasmid were constructed. The vectors named pIGDMCT7RS, pIGDM4RS and pIGDMKAN carry various antibiotic resistance genes: chloramphenicol, ampicillin or kanamycin, respectively. Two derivatives contain the inducible T7 promoter while the third one bears the constitutive pms promoter from a clinical strain of Klebsiella pneumoniae. The pIGDM1-derivatives are compatible with other ColE1-like plasmids commonly used in molecular cloning. The pIGDMCT7RS and pIGDM4RS vectors contain genes encoding AGA and AGG tRNAs, which supplement the shortage of these tRNAs, increasing the efficiency of synthesis of heterologous proteins. In conclusion, pIGDMCT7RS, pIGDM4RS and pIGDMKAN vectors, with significantly improved features, including compatibility with vast majority of other plasmids, were designed and constructed. They enable a high-level expression of a desired recombinant gene and therefore constitute a potential, valuable tool for pharmaceutical companies and research laboratories for their own research or for the production of recombinant biopharmaceuticals.

The design and fabrication of a continuous-flow μPCR device with very short amplification time and low power consumption are presented. Commercially available, 4-layer printed circuit board (PCB) substrates are employed, with in-house designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network, where DNA amplification takes place. The 1.9-m-long microchannel in combination with desirably high flow velocities (for fast amplification) challenged the robustness of the sealing that was overcome with the development of a novel bonding method rendering the microdevice robust even at extreme pressure drops (12 bars). The proposed fabrication methods are PCB compatible, allowing for mass and reliable production of the μPCR device in the established PCB industry. The μPCR chip was successfully validated during the amplification of two different DNA fragments (and with different target DNA copies) corresponding to the exon 20 of the BRCA1 gene, and to the plasmid pBR322, a commonly used cloning vector in E. coli. Successful DNA amplification was demonstrated at total reaction times down to 2 min, with a power consumption of 2.7 W, rendering the presented μPCR one of the fastest and lowest power-consuming devices, suitable for implementation in low-resource settings. Detailed numerical calculations of the DNA residence time distributions, within an acceptable temperature range for denaturation, annealing, and extension, performed for the first time in the literature, provide useful information regarding the actual on-chip PCR protocol and justify the maximum volumetric flow rate for successful DNA amplification. The calculations indicate that the shortest amplification time is achieved when the device is operated at its enzyme kinetic limit (i.e., extension rate). Graphical abstract.

The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the noncanonical homology-directed DNA repair (HDR) based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the short-homology-mediated genome editing and found that the HDR pathway provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing short homology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

The Drosophila transgenic technology and fluorescent protein fusions are powerful tools to analyze protein expression patterns, subcellular localization and protein dynamics. Recently, the Drosophila transgenic technology has been improved by the highly efficient phiC31 site-specific integration system. Many new and improved fluorescent proteins with desirable advantages have been developed. However, the phiC31 system and the newly developed fluorescent proteins have not been systematically applied in Drosophila transgenic vectors. Here, we have constructed a modular toolset of C-terminal fluorescent protein fusion vectors based on phiC31 site-specific integration system for the generation of transgenic Drosophila lines. These cloning vectors contain a variety of fluorescent tags, including blue, cyan, green or red fluorescent proteins, photoactivatable or photoswitchable fluorescent proteins, fluorescent timers, photosensitizers and bimolecular fluorescence complementation tags. These vectors provide a range of transcriptional regulation options including UAST, UASP, UASC, LexAop, QUAS, Ubi, αTub67C and αTub84B promoters, and two screening marker options including white and vermilion gene. The vectors have been tested in vivo and can produce fluorescent chimeric proteins that are functional.

Carbon dioxide (CO2) is one of the main causes for global warming. The most important enzyme that converts CO2 into bicarbonate and prevents CO2 emissions in the environment is carbonic anhydrase (CA). The aim of this study was to clone the heat-stable CA gene in Escherichia coli host. The CA gene coding frame of Caminibacter mediatlanticus was optimized based on the more frequently used codons by E. coli. Accordingly, nine codons were inserted in this gene fragment. After CA gene insertion in cloning vector (pGH), it was sub-cloned in pBI121 expression vector and transformed into E. coli XL1-Blue. The accuracy of gene cloning was confirmed by PCR, restriction enzyme digestion and sequencing. Cell extract was prepared from recombinant bacteria. Culture supernatants were assayed for CA activity by the color change of bromothymol blue as indicator. The time for the indicator color changes were recorded and the assays were repeated five times. The average of color change time for cell extract of recombinant, non-recombinant E. coli and control reaction were 1.5, 16, and 48 min, respectively. The amount of active enzyme was calculated to be 92.8 units/ml and the enzyme retained its activity after of incubation at 60 °C, 65 °C, and 70 °C. Due to the positive properties of CA from C. mediatlanticus, codon optimization, modification, and expression of this gene, using a simple and inexpensive method, may be used to obtain CA with enhanced properties.

With the passage of time, energy sources are decreasing day by day. In order to meet the world's demand, much attention is being paid to the study of enzymes with xylanolytic activity as a potential means of generating energy. A thermophilic xylanolytic bacterium, Bacillus sp., was isolated from naturally decaying material by enrichment culture and serial dilution methods. The bacterium was grown in MH medium at 50°C and pH 7 for 10 h. The xylanolytic Bacillus sp. produced clear yellow haloes around the colonies in the presence of p-nitrophenyl beta-D-xylopyranoside (pNPX) as a substrate. After condition optimization, it was found that the organism produced the higher level of xylosidase activity after 14 h in the presence of arabinose as a carbon source and ammonium sulfate as a nitrogen source in the pH 7 medium of at 55°C. The maximum β-xylosidase activity after optimizing the culture condition was 5.0 U/mL. Later this thermophilic Bacillus sp. was used as a donor in cloning of the β-xylosidase gene. A genomic library of Bacillus sp. was prepared by digesting the genomic DNA of the Bacillus with the restriction endonuclease BamHI, ligating the fragments in the pUC18 cloning vector and then transforming the competent E. coli DH5α cells with the resultant chimeric plasmid. The β-xylosidase gene was identified by screening the transformants in duplicates on LB agar plates overlaid with pNPX as a substrate. Commercial production of β-xylosidase to be used as a methanol-producing enzyme can help to overcome fuel shortages.

A gene encoding chitinase from B. subtilis has been isolated after optimization of PCR conditions. It was cloned with two different prometers, T7 promoter of the pJET1.2/blunt cloning vector and the SP6 promoter of pGEM®-T Easy vector. After transforming E. coli DH5α, two transformants were selected, CHI-NRC-4 from the first vector and T-CHI-NRC-6 from the second vector, and used for further studies. The complete CDS sequence of chitinase gene was determined and submitted to GenBank with the accession number KX268692.1. Culture supernatants of E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed up to 96% inhibition in egg hatching due to both E. coli transformants as compared to control which reflect the same expression efficiency of both used prometers. A greenhouse experiment was carried out to evaluate the nematicidal effect of culture supernatants of the two transformts E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control. Keywords: Gene cloning, Chitinase, Nematode, Meloidogyne javanica, Eggplant

Brucellosis is a highly contagious and zoonotic disease and has a considerable impact on animal health and economy of a country, principally in Pakistan, where rural income largely depends upon livestock farming and dairy products. The disease burden is more in underdeveloped/developing countries due to the low economy and limited access to the diagnostic facilities. In Pakistan, the prevalence of Brucella abortus is very high, so it is the need of the hour to control this disease through more advanced methods. This study was designed with the aim to construct the DNA based vaccine of gene encoding antigenic surface protein (BCSP31). For this purpose, the BCSP31 gene was amplified, purified and ligated in pTZ57 R/T (cloning vector). Dubbed BCSP31-pTZ57 R/T vector was transformed into competent cells (DH5α). After plasmid extraction, the plasmid and pET-28a vector was restricted with EcoRI and BamHI. Again, ligation was done and dubbed pET-28a-BCSP31 transformed into E. coli (BL21). After expression, the protein was purified and used for evaluation of immunogenic response. The protective and immunogenic efficacy of the vaccine was evaluated in rabbits (n = 20). The rabbits were divided into four equal groups. Groups A-C were given purified protein diluted in normal saline @ 750, 1500 and 3000 μg/0.2 mL, respectively through intraconjunctival route. Group D was given 0.2 mL normal saline through intraconjunctival route. Specific immunoglobulin G (IgG) responses were measured through indirect ELISA on a weekly basis. The titer of IgG against the antigen was significantly (p < 0.05) higher in vaccinated groups A-C as compared to group D (control group) in a dose dependent manner. Moreover, log units of protection produced by DNA based vaccine in the rabbits (3.02) also indicated the protective efficacy of the DNA vaccine against B. abortus challenge. The response of this vaccine in rabbit suggested its potential effectiveness against Brucella abortus in large animals.