Figure 3 - available via license: Creative Commons Attribution 3.0 Unported
Content may be subject to copyright.
![Electrophoresis results of protein expression products using polyacrilamide gel with a voltage of 100 volts and Comassie blue coloring 0.1%. [M]: Protein ladder marker; [(+)]: Positive controls; [(-)]: Negative controls; [C1a]: C1a clone that has expression.](publication/337364990/figure/fig3/AS:827001648709632@1574183966071/Electrophoresis-results-of-protein-expression-products-using-polyacrilamide-gel-with-a.png)
Electrophoresis results of protein expression products using polyacrilamide gel with a voltage of 100 volts and Comassie blue coloring 0.1%. [M]: Protein ladder marker; [(+)]: Positive controls; [(-)]: Negative controls; [C1a]: C1a clone that has expression.
Source publication
This research aimed to express F recombinant protein that is clon from genes F of local isolate ND virus which can be used as vaccine candidate in order to improve the effectiveness of ND virus vaccination. Confirmation of NDV pBT7-N-His-Fusion plasmid on C1a clone is done by gel agarose 1 % electrophoresis with staining by using fluorosafe DNA sta...
Contexts in source publication
Context 1
... find out the results of protein expression, a polyacrylamide gel electrophoresis (PAGE) was confirmed. Electrophoresis results of protein expression products are shown in Figure 3. Proteins separate into bands according to their molecular weight. ...
Context 2
... and negative controls are used as a determinant of the presence or absence of protein F which is a sign of success of the protein expression process. The visualization of polyacrylamide gel with coomassie brilliant blue (CBB) staining in Figure 3. shows that in the C1a well there was a protein band of 25.6 kDa after being compared with protein markers and positive control showed a 28 kDa protein band. ...
Citations
... In this work, the recombinant F protein of NDV had successfully in-vivo expressed into E. coli BL21(DE3) as a protein fragment with a molecular weight of 25.6 kDa under an optimal condition by 1.0 mM IPTG induction when the OD600 of the cell was 0.6 and an induction duration of 8 h. The results of this study were in line with Putri and Haryanto (2019), who carried out the in-vitro expression of NDV recombinant F protein from E. coli C-1a clone and Haryanto et al. (2020), who successfully conducted in-vitro expression of the recombinant F protein of NDV from local Indonesian isolates by using a cell-free protein expression system. This finding also was concurrent with another researcher group Astuti et al. (2020), who have successfully expressed and purify the recombinant F protein of local isolate NDV and studied the antibody response to recombinant F protein in broiler chicken post-vaccination. ...
Wulanjati MP, Witasari LD, Wijayanti N, Haryanto A. 2021. Recombinant fusion protein expression of Indonesian isolate Newcastle disease virus in Escherichia coli BL21(DE3). Biodiversitas 22: 3249-3255. Newcastle disease is a major problem in poultry industry due to high mortality of susceptible chicken. New vaccine agents are important to be developed to eliminate the disease threat. This study aimed to examine the expression of fusion (F) protein of Newcastle Disease Virus (NDV) from Indonesian isolates in E. coli BL21(DE3) by IPTG induction. The sample was a part of F gene of NDV from Galur, Kulon Progo, Yogyakarta, Indonesia (0663/04/2013) with a molecular size of 600 bp that was synthesized and inserted into pBT7-N-His expression vector. The recombinant F protein with molecular weight of 25.6 kDa was successfully expressed in E. coli BL21(DE3), purified using Ni-NTA magnetic silica beads, and confirmed by western blotting. Optimization of expression showed that recombinant F protein was optimally expressed by induction of 1.0 mM IPTG when the cells reached OD600 = 0.6. The induction duration was 8 h. B-cell epitopes prediction showed that F protein possessed four epitopes that possibly recognized by B-cell. Since recombinant F protein was considered to possess immunogenicity, its potency as a candidate of NDV vaccine agent should be investigated in the future.
... The re sult of this work showed that the recombinant F protein of NDV was successfully expressed by a cellfree protein ex pression system and visualized by using SDSPAGE elec trophoresis with Coomassie Brilliant Blue staining and Western blot as a specific protein with a molecular weight of 25.6 kDa. This result was in line with the recombinant protein study performed by other researchers, who have successfully expressed the recombinant F protein of NDV from E. coli clone of C1a (Putri and Haryanto 2019). ...
The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV). The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.
Dengue Hemorrhagic Fever (DHF) is a disease generated by dengue virus infection. The dengue virus is transmitted to the host by mosquito vectors. The main and secondary vectors are Aedes aegypti and Aedes albopictus. Dengue virus transmission is mediated by salivary gland proteins that facilitate the blood-feeding process. Previous studies have shown that the molecular weight of 31 kDa is an immunogenic protein of Ae. aegypti belonging to the D7 family based on the result of Mass Spectrometry. This immunogenic protein can be used for the development of vector-based dengue vaccines. The purpose of this study is to purify the 31 kDa protein fraction from the salivary gland of Ae. aegypti and Ae. albopictus using the electroelution method. The study is conducted by collecting the salivary glands of Ae. aegypti and Ae. albopictus, SDS-PAGE, electroelution using the Bio-Rad 422 electroeluter, Dot Blot, and Western Blot. Both protein samples resulting from electroelution confirmed with a single band appeared in SDS-PAGE. The optimal conditions for electroelution are 3 h running time, 100 voltage, volume ±1 mL, and the concentration obtained are 1.789 mg/mL (Ae. aegypti) and 1.81 mg/mL (Ae. albopictus). These results are supported by the other result of the dark dots shown in Dot Blot and a single band of 31 kDa shown in Western Blot when it reacted with the serum of dengue patients, endemic healthy people, and neonates. These results indicate that the purified 31 kDa immunogenic protein fraction can be recognized by specific antibodies.
Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli , then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C -2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..
