Protein Banding Patterns Research Articles

Protein profiling for phylogenetic relationship in snakehead species

Protein banding pattern of eight snakeheads – Channa species viz., Channa striatus, Channa marulius, Channa punctatus, Channa diplogramme, Channa bleheri, Channa gachua, Channa stewartii and Channa aurantimaculata collected from different regions of India were used to study the phylogenetic relationship among them. The banding pattern from muscle protein indicated a unique profile for each species and the electrophoregrams showed similarities among the species studied. In the SDS-PAGE, a maximum of 12 protein bands were obtained for C. gachua followed by 11 for C. diplogramme and 10 for C. marulius whereas less number of bands were recorded for the remaining species. Molecular weight of the protein bands varied from 16 kDa - 232 kDa. UPGMA (Unweighted Pair Group Method with Arithmetic Mean) dendrogram revealed that the phylogenetic relationship was very close among C. aurantimaculata and C. bleheri and also between C. gachua and C. stewarti.

Venom phenotypes of the Rock Rattlesnake (Crotalus lepidus) and the Ridge-nosed Rattlesnake (Crotalus willardi) from México and the United States

Although the Mexican Highlands has the highest diversity of small-bodied rattlesnakes in the world, studies on the species found throughout this region have been relatively scarce. This has led to challenges with examining venom phenotypic characteristics, as well as species misidentifications and misclassifications. In the current study we investigated venom variation among four subspecies of Crotalus lepidus (C. l. klaluberi, C. l. lepidus, C. l. maculosus, C. l. morulus) and four subspecies of C. willardi (C. w. amabilis, C. w. obscurus, C. w. silus, and C. w. willardi) that inhabit regions of southwestern United States and central México. SDS-PAGE patterns show the presence of many of the major compounds found in other rattlesnake venoms, although minor variations in protein banding patterns and intensity are recognizable. Most notably, PI-metalloproteinase (SVMP) bands appear to be very faint to absent in northern C. l. lepidus and C. l. klauberi subspecies, but are fairly prominent in all other C. lepidus and C. willardi subspecies. Enzyme activity assays revealed that C. lepidus subspecies exhibit higher SVMP and thrombin-like activities when compared to C. willardi subspecies. Significant differences between subspecies were also observed for kallikrein-like serine protease, L-amino acid oxidase, and phosphodiesterase activities, although these differences appear to be random and fail to follow a geographical or phylogenetic trend. The same relationship was also observed for fibrinogenolytic and coagulation assays. Toxicity assays conducted on lab mice (Mus musculus), house geckos (Hemidactylus frenatus), and house crickets (Acheta domestica) revealed varying toxicities between subspecies, with C. l klauberi being the most toxic towards mice (LD50 = 1.36 μg/g) and house geckos (LD50 = 0.17 μg/g), and C. w. silus being most toxic to house crickets (LD50 = 1.94 μg/g). These results provide additional evidence that geographical isolation, natural selection, and adaptive evolution in response to diets may be driving forces contributing to population-level variation in venom composition.

English

Contamination of soil with heavy metals is one of the major environmental problems in many countries that reach from many sources as power stations, application of metal pesticides, fertilizers, and sewage sludge. In this study, ten Rhizobium leguminosarum bv. viciae isolates were collected from different governorates in Egypt (Menoufia, Kafr El-Sheikh, Qalubia, Fayoum, Ismailia, Sharkya, Dakhalia, Behira and North Sinai) to evaluate their resistance to three heavy metals (Cu, Zn and Pb). The results showed that the isolates of RL3 and RL6 exhibited the best resistance toward the heavy metals tested. PCR based specific primers were used to screen the tested isolates for detection of some heavy metal resistant genes (copA, pbrA and czcD). The Pb-resistant gene pbrA was detected in most of tested isolates except RL7 and RL9 isolates, while, the Cu-resistant gene copA was found in all isolates except RL1, RL2, RL4 and RL8 isolates; however, the Zn-resistant gene czcD was detected only in the RL9 isolate. SDS-PAGE analysis was used to study the protein banding patterns for some tested isolates under lead stress and compared them with their untreated control. Key words: CopA, pbrA and czcD genes, heavy metals, Rhizobium.

Grain protein estimation and SDS-PAGE profilingof six important arid legumes

Grain protein content and protein profiles of six arid grain legumes belonging to three different tribes were studied. Average grain protein (per cent) of horse gram, cowpea, moth bean, guar, chickpea and green gram was 20.29, 22.8, 22.6, 20.6, 21.82, and 20.40 respectively. All the six arid legumes were clearly differentiated based on the protein banding patterns generated through SDS-PAGE. The molecular weight range of proteins in different legumes was observed to be ~16 to ~250kD. Interestingly, in chickpea, moth bean and horse gram proteins >100kD were not mostly observed. Further, distinct proteins were observed indicating inter-varietal differences in horse gram (CRHG-19), cowpea (RC-101) and guar (HG-2-20), which could be useful in varietal identification. The different legumes were broadly clustered in to four clusters. Further, chickpea (Cicereae), green gram and cowpea (Phaseoleae) were clustered together. The similarity indexes of varieties and individual crops ranged from 0.667 to 0.950 and 0.425 to 0.821 respectively. Though the six legumes belong to Leguminosae they have distinct protein profiles which could be useful in species/varietal identification.

Gene Over-Expression of Glycerol-3-phosphate Dehydrogenase in Some Marine Algae of Egypt.

Studies focusing a survey or a comparison between freshwater and marine algae are relatively Algae exhibit a wide range of acclimation in response to induced salinity. However, studies focusing a survey or a comparison between freshwater and marine algae are relatively scarce. A comparative study between three fresh water algae (Spirogyra sp., Chara sp. and Cladophora sp.) and six marine algae among them brown: Sargassum dentifolium, Padina boryana, Dictyota dichotoma; red: Gelidium latifolium, Gracilaria dura and green: Enteromorpha intestinalis) has been performed to study how marine algae can cope with salinity of seawater. Algal fluorescence from the marine and fresh water groups was analyzed to determine whether differences in room-temperature fluorescence could be ascribed to differences between algal classes or specific for individuals. Results obtained showed that fluorescence signatures were relatively uniform for species within class but were different for different classes. Scanning of the SDS-gel showed the presence of certain characteristic protein banding pattern for each species. The amount of proline showed irregular differences according to species. Mannitol was detected only in the members of brown algae. Moreover, the mineral (sodium and potassium) and glycerol contents of the marine algae has noticeable greater values than those of fresh water members. The expression level of G3PDH mRNA was significantly upregulated in marine algal group as compared to that of the freshwater algae. GPDH had a significant impact on numerous metabolic processes in marine algae, including the biosynthesis of glycerol.

Identification and characterization of Memecylon species using isozyme profiling

Background:The protein/isozyme fingerprint is useful in differentiating the species and acts as a biochemical marker for identification and systematic studies of medicinal plant species.Objective:In the present study, protein and isozyme profiles for peroxidase, esterase, acid phosphatase, polyphenol oxidase, alcohol dehydrogenase, and alkaline phosphatase of five species of Memecylon (Melastomataceae), Memecylon umbellatum, Memecylon edule, Memecylon talbotianum, Memecylon malabaricum, and Memecylon wightii were investigated.Materials and Methods:Fresh leaves were used to prepare crude enzyme extract for analyzing the five enzymes isozyme variations. Separation of isozymes was carried out using polyacrylamide gel electrophoresis (PAGE) and the banding patterns of protein were scored. Pair-wise comparisons of genotypes, based on the presence or absence of unique and shared polymorphic products, were used to regenerate similarity coefficients. The similarity coefficients were then used to construct dendrograms, using the unweighted pair group method with arithmetic averages.Results:A total of 50 bands with various Rf values and molecular weight were obtained through PAGE analysis. Among the five Memecylon species, more number of bands was produced in M. wightii and less number of bands was observed in M. edule. The results of similarity indices grouped M. malabaricum and M. wightii in one cluster with 98% similarity and M. umbellatum, M. edule, and M. talbotianum are grouped in another cluster with 79% similarity showing close genetic similarities which is in accordance with the morphological identification of Memecylon species.Conclusion:The protein/isozyme fingerprint is useful in differentiating the species and acts as a biochemical marker for identification of Memecylon species.SUMMARY Biochemical characterization of Memecylon species was evaluated by SDS-PAGE of extracted protein and isozyme profiling on native PAGE.After electrophoresis, each gel was stained with specific stains. Genetic distance relationships were evaluated based on the banding patterns of protein on isozymes.Unique banding pattern of esterase, peroxidase, acid phosphatase, alcohol dehydrogenase and polyphenol oxidase are observed in all the five species of Memecylon, which represent the fingerprint of Memecylon species.SDS-PAGE and isozyme profiling of five Memecylon species revealed that M. malabaricum and M. wightii grouped in one cluster and M. umbellatum, M. edule and M. talbotianum grouped in another cluster showing close genetic similarities which is in accordance with the morphological identification of Memecylon species.This is the first report on the comparison of protein and isozyme profile of five different Memecylon species. Abbreviations Used: SDS-PAGE: Sodium docecyl sulfate polyacrylamide gel electrophoresis; NTSYS PC2: Numerical taxonomy system, version 2.2 for Windows XP, Vista, Win7, Win 8 and Win10 including 64 bit

Ground Nut Research in Odisha, A Case Study and Analysis

Ground Nut Research in Odisha, A Case Study and Analysis

Electrophoretic Analysis of Salivary Gland Proteins of Adult Culex antennatus (Diptera: Culicidae)

Salivary glands of mosquitoes play an important role in food ingestion and digestion as well as transmission of pathogens. Mosquitoes are able to adapt to feed on blood by salivary glands which secrete proteins that work against the haemostasis process. So, the identification of these protein contents in the salivary glands of Culex antennatus (males and females) is the aim of this study. In the present study, protein banding pattern (Native and SDS PAGE) of salivary glands of adult Culex antennatus (un-fed, sugar-fed, starved and blood-fed) was investigated. Males and females of Cx. antennatus were dissected and their salivary glands were collected at un-fed stage, 0-, 3-, 6-, 12- and 24- h after sugar feeding and starved stage. Female salivary glands were additionally collected at different stages of blood feeding (probing time, partial engorgement, full engorgement, 3-, 6-, 12-, 24-, 48- and 72- h. after blood meal and after oviposition. Results of native-PAGE demonstrated that there were differences in the overall protein banding pattern in salivary glands of males and females of Cx. antennatus in the cases of un-fed, sugar-fed and starved stages. Differences in salivary gland proteins were observed when comparing males of all stages, too. Differences in salivary gland proteins were also noticed when comparing females of all stages. Furthermore, all blood-feeding stages of females showed differences in salivary gland proteins when compared to sugar-fed and starved females. Results of SDS-PAGE clarified that the molecular weight of the separated proteins (in all stages) ranged from 317.36 to 10.91 KDa. Differences were also observed between males and females in the cases of un-fed, sugar-fed and starved stages of Cx. antennatus.

Comparative study on the biosorption of aluminum by free and immobilized cells of Bacillus safensis KTSMBNL 26 isolated from explosive contaminated soil

The study describes the sorption of aluminum by Bacillus safensis isolated from an explosive contaminate site. In a batch system, the optimum pH, temperature and contact time on biosorption of Al and cell growth were found to be 6.0, 35°C and 24h, respectively with initial metal concentration of 100mg/l Al. In immobilization studies, sodium alginate was used as supporting material for cell entrapment under optimized conditions. The variation in the protein banding pattern, functional groups and nature of Al treated and untreated bacterial cells were studied using SDS-PAGE, FTIR and XRD. While comparing the results of batch and immobilized sorption studies, the metal removal efficiency of free cells (92mg/l) was slightly higher than the immobilized cells (84mg/l). But the maintenance of high cell density, low processing cost, repeated and continuous uses are all recommends the immobilization technique for large scale water treatment.

Profenofos induced modulation in physiological indices, genomic template stability and protein banding patterns of Anabaena sp. PCC 7120

To understand the mechanism underlying organophosphate pesticide toxicity, cyanobacterium Anabaena PCC 7120 was subjected to varied concentrations (0, 5, 10, 20 and 30 mg L−1) of profenofos and the effects were investigated in terms of changes in cellular physiology, genomic template stability and protein expression pattern. The supplementation of profenofos reduced the growth, total pigment content and photosynthetic efficiency of the test organism in a dose dependent manner with maximum toxic effect at 30 mg L−1. The high fluorescence intensity of 2′, 7′ –dichlorofluorescin diacetate and increased production of malondialdehyde confirmed the prevalence of acute oxidative stress condition inside the cells of the cyanobacterium. Rapid amplified polymorphic DNA (RAPD) fingerprinting and SDS-PAGE analyses showed a significant alteration in the banding patterns of DNA and proteins respectively. A marked increase in superoxide dismutase, catalase, peroxidase activity and a concomitant reduction in glutathione content indicated their possible role in supporting the growth of Anabaena 7120 up to 20 mg L−1. These findings suggest that the uncontrolled use of profenofos in the agricultural fields may not only lead to the destruction of the cyanobacterial population, but it would also disturb the nutrient dynamics and energy flow.

ANALISIS KANDUNGAN SAPONIN PADA DAUN DAN TANGKAI DAUN Carica pubescens Lenne & K. Koch

<p><em>Carica pubescens Lenne & K. Koch is a tropical species that adapt to the plateau environment and low temperatures. In East Java, the plant is found in Cangar and Bromo region. Morphological, chemical content, and analysis of protein banding pattern on C. pubescens has been done, but more on the analysis of active compounds for pharmaceutical raw materials and its accumulation in the body of the plant has not been widely studied. Saponins on C. pubescens potential as a raw material of natural medicine in the treatment of Diabetes Mellitus (DM). This study aims to determine the content of saponin in leaf and petiole of C. pubescens in terms of absorbance values. Saponins were analyzed by qualitative form the foam test, color test, Thin Layer Chromatography (TLC) analytical and preparative. Quantitative test in the form of UV-Vis spectrophotometry results preparative TLC. This research was done at the Laboratory of Department of Biology and Chemistry UIN Maulana Malik Ibrahim Malang. The results showed that the leaf and petiole of C. pubescens positive for the saponins with the formation of stable foam for 60 seconds at 1.5 cm - 1.7 cm. The third positive samples containing saponins triterpene the ring test produces a brownish color. Isolation saponin by TLC shows the best ratio of eluent chloroform: methanol: water (14: 6: 1) compounds can be separated perfectly. Saponin absorbance values obtained three samples as follows: petiole samples from the region Cangar amounted to 0.852, leaf samples from the region Cangar amounted to 0.686, and leaf samples from Bromo region amounted to 0,629. The highest saponins found in organs petiole. Thus the petiole of C. pubescens has the potential to be used as a source of triterpene saponins which can be developed into a commercial herbal medicines.</em></p>

IDENTIFIKASI SENYAWA ANTIDIABETES SECARA in Silico PADA Carica pubescens Lenne & K. Koch

<em>Carica pubescens Lenne & K. Koch is one of the typical highland crops in Indonesia. This plant can be found in the region of Cangar and Bromo in East Java, as well as the Dieng Plateau, Central Java Wonosobo-Banjarnegara. Morphological, chemical content, and analysis of protein banding pattern of the Carica pubescens has been done, but more about the use of active compounds for pharmaceutical raw materials, in Diabetes Mellitus type 2 have not been studied. One therapeutic approach to lowering blood sugar levels posprandial is to slow down or inhibit the absorption of glucose through inhibition of the carbohydrates hidrolitic enzyme such as α-amylase and α-glucosidase. This study aims to identify antidiabetic compounds in silico. Antidiabetic compounds were analyzed by methods of docking. The results showed that papain and cysteine protease in C. pubescens have better binding affinity to α-amylase and α-glucosidase compared to iridoid glycosides. Papain and cysteine proteases isolated from C. pubescens are potential to be developed as an antidiabetic.</em>

Copper and Zinc Interactions with Cellular Prion Proteins Change Solubility of Full-Length Glycosylated Isoforms and Induce the Occurrence of Heterogeneous Phenotypes.

Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrPSc), which result from the conformational conversion of physiological prion proteins (PrPC). PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC–Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays.

Evaluation of genetic diversity in some promising varieties of lentil using karyological characters and protein profiling.

Somatic chromosome study from root tip cells using the squash technique of the cytological method and seed protein profile of 5 varieties of Lens culinaris (Lentil) through SDS–PAGE were investigated. Karyotype analysis showed gross uniformity in morphology. Somatic chromosome number 2n = 14 is constant for all the varieties. Chromosomes are mostly long to medium in length with secondary constrictions in one pair of chromosome. Primary constrictions in chromosome ranged from nearly median to nearly submedian in most of the cases. Notwithstanding the gross homogeneity, karyotype analysis revealed minute differences in details. Each lentil variety is thus characterized by its own karyotype, serving as one of the identifying criteria. The seed protein profile revealed that varieties are very close to each other with respect to similarity index that ranged from 0.594 to 0.690. With regard to seed protein banding patterns, slight polymorphism (14.285%) indicating low genetic diversity has been identified among the 5 varieties. A dendrogram indicates one variety is plesiomorphic and rest varieties are apomorphic. All the experimental varieties of lentil studied here show low genetic diversity due to their similar genetic base, indigenous genetic resources and conservative nature of the seed protein.

Analysis of seed storage protein diversity among twelve different cultivars of Pea (Pissum sativum L.) using SDS-PAGE.

The present investigation was carried out during 2012-13 with twelve Pea ( Pisum sativum L.) cultivar for protein profiling through SDS-PAGE in The department of Genetics and Plant Breeding, CSA University of Agriculture and Technology, Kanpur (U.P). The Seed protein profiles of pea cultivars were studied by extracting the total proteins from five single seeds in each cultivar and performed SDS-Polyacrylamide gel electrophoresis. All the cultivars were clearly revealed remarkable polymorphism from their protein banding patterns. On the basis of banding patterns through SDS-PAGE, indicated that the number of bands found in cultivars ranged from 12 to 19 with Rm value 0.12 to 0.9. The results found that, the cultivar KPMR-400 had recorded highest number of bands (19) whereas, the minimum number of bands (12) observed in three cultivars viz., KPMR-921, KPMR-902 and KPMR-913. The total seed protein variation were also analyzed using Un-weighted Pair Group Method with Arithmetic Mean (UPGMA) and resultant cluster analysis based on the data of protein profiling, classified twelve cultivars into six major groups. The finally study concluded that, the protein variability analysis clearly showed that there was sufficient genetic divergence among these cultivars of pea with respect to seed storage protein. Among all the cultivars, the KPMR-906 in cluster IV having wider genetic diversity and suggested to utilize in future crop improvement program.

Caspase-12 as a Biomarker of Aquatic Pollution in Oreochromis niloticus

Biomarkers have recently been used in studies of aquatic environment. Here we present data on stress related bio-markers in Oreochromis niloticus (Nile tilapia) as indicators of aquatic pollution. The samples of study were collected from contaminated aquatic habitats (Qaroun Lake and its neighboring fish farm), in comparison with the reference site. Heavy metal concentrations as Fe, Mn, Zn, Cu and Cd were determined in the water and the fish tissues. Oxidative stress biomarkers (Malondialdehyde, reduced glutathione, glutathione S transferase and catalase) together with endoplasmic reticulum stress biomarker (Caspase-12) were determined in both the blasma and the tissue samples. Histopathological examination of kidney and muscles with SDS electrophoresis of muscle proteins were used to study the influence of pollution on Nile tilapia. Heavy metal concentrations of water and tissue samples from contaminated habitats were significantly higher than the reference site. A highly significant difference in stress related biomarkers was recorded in the fish from contaminated habitats. Histopathological modulations and disrepairs were observed in fish tissues collected from contaminated habitats. Results have shown differences in banding pattern of muscle proteins of Nile tilapia collected from polluted sites. These results provide evidence that caspase-12 is a useful biomarker for aquatic pollutions.

KAJIAN PENGARUH VARIASI KONSENTRASI ASAM SITRAT TERHADAP KEKUATAN GEL PRODUK GELATIN KULIT AYAM BROILER DIKAITKAN DENGAN POLA PROTEINNYA

Gelatin is a biopolymer that can be generated from partially hydrolysis of collagen tissue. Extraction of gelatin consists of pretreatment and thermal extraction steps. Pretreatment process used sodium hydroxide to remove non collagen protein in matrix sample, sulfuric acid to demineralize, and citric acid to hydrolyse. The aim of this research was to study the effect of variation in concentrations of citric acid used in hydrolysis process on the gel strength and protein profile of gelatin products extracted from broiler chicken skin. The variation of the concentration citric acid used was 0.7 % (GA); 1.5% (GB); and 3.0% b/v (GC) respectively. The gel strength was measured using CT3 Texture Analyzer and protein profile of gelatin product was analyzed by SDS-PAGE method. The result showed that variation in concentration of citric acid used in the pretreatment process affected the gel strength and protein profile of gelatin product. Increasing the concentration of citric acid used in pretreatment process decreased the gel strength and molecular weight of gelatin product. Gel strength of each gelatin product was 265.81 g bloom for GA ; 196.05 g bloom for GB (1.5%), and 35.32 g bloom for GC (3.0 %) respectively. The electropherogram of both GA (0.7%) and GB (1.5%) revealed similar pattern of protein bands but the thickness of each bands was different. On the other hands, GC (3.0%) did not show any protein bands on the eletropherogram. The best gelatin product obtained in this experiment was found by using 0.7 % b/v citric acid (GA) in the pretreatment process. The gelatin product (GA) had characteristics as follows: yield 15.73%; moisture 7.30%; ash 0.51%; protein content 97.95%; fat content 0.62%; gel strength 265. 81 g bloom and thicker protein bands than others.

Effect of nonthermal plasma on physico-chemical, amino acid composition, pasting and protein characteristics of short and long grain rice flour

Nonthermal plasma (NTP) is a novel technology with potential applications in food property and functionality modification. In this study, short and long grain rice flours were exposed to different plasma intensities and resulting ozone concentrations, and their physicochemical, amino acid composition, X-ray diffraction, pasting properties and protein profile were evaluated. The L*, b*, blue value, λmax, pH, transmittance, swelling power, and syneresis of both rice types increased with NTP treatment while a* decreased. X-ray diffraction revealed a decrease in crystallinity with NTP treatment. Long grain rice showed higher peak viscosity, breakdown viscosity and lower pasting temperature as compared to short grain rice due to higher amylose content. Glutamic acid, asparagine, serine histidine, threonine, γ-aminobutyric acid, tryptophan, isoleucine, phenylalanine and proline content increased with NTP treatment in both rice types. SDS page analysis did not reveal any effect of NTP treatment on banding pattern of total protein of both rice types.

EFFECT OF INFESTATIONWITH GLADIOLUS THRIPS, THRIPS SIMPLEX (MORISON) ON THEVASE LIFE PERIOD OF GLADIOLUS FLOWERS

experiments were carried out to study the effect of insect infestation by gladiolus thrips, Thrips simplex ( Morison ) on the vase life period of gladiolus flowers after picking under glasshouses condition and flowers contents specifically two elements (sugar and protein) which correlate with vase life period. Morevore effect of infestation with this insect on the interior tissues of the gladiolus flowers petals was also conducted. Experiments were done in El-Orman garden, Giza Governorate throughout season 2014. The vase life period of the infected gladiolus flowers withT.simplex ranged from between 3-5 days while the vase life period of non-infected flowers ranged between of 7-9 days. Results indicated that the total soluble sugar and total protein were significantly lowerin different flowercolures of gladiolus (yellow, red, pink and white) infested with gladiolus thrips, T. simplex compared with control. Also, results showed that there werechanges in protein banding patterns in petals of gladiolus flowers corresponded with infectionby T. simplex.

Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae.

Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although three lepidopteran insect larvae (Helicoverpa armigera, Spodoptera litura and Samia cynthia ricini) were tested for production of VLPs, expression was obtained only in Eri silkworm larvae. High titred recombinant baculovirus encoding the polyprotein P1-2A-3C of FMDV was prepared in Sf9 cells. Injection of recombinant baculovirus into hemocoel of Eri silkworm larvae resulted in increasing levels of expression of VLPs in the hemolymph from 3 to 7days post infection (dpi) compared to low level expression by oral feeding. The VLPs reacted in Sandwich ELISA with serum raised against whole virus particles of FMDV type O/IND/R2/75 and protein banding pattern of 26, 37 and 47kDa in Western blotting demonstrated their antigenic resemblance to native virus. Sucrose density gradient purified VLPs were used for immunization of rabbits and guinea pigs for assessing immunogenicity. Further, the reactivity of serum samples of rabbits and guinea pigs in Indirect-ELISA with titres (1.30-2.81 Log10) indicated that the VLPs were antigenic and immunogenic in nature. We demonstrate that Eri silkworm larvae could be used for production of VLPs of FMDV type O/IND/R2/75 for the first time. This approach could be useful for large scale production of recombinant VLPs for vaccine or diagnostic use in FMD control programme.