In vitro expression of the recombinant fusion protein of Newcastle disease virus from local Indonesian isolates by using a cell-free protein expression system

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 Escherichia 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 andWestern 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.


Introduction
Newcastle disease (ND) is a poultry infectious viral dis ease which has become a major problem in the poultry in dustry in developing countries. Office International des Epizooties (OIE) categorizes ND in the A list of animal diseases, which includes infectious diseases with rapid and serious spreading and affecting public health and socio economic communities ( [OIE] Office Internationalof Epi zootics 2015). As other infectious viral disease outbreak which had been reported in Indonesia poultry industry, such as avian influenza (Njoto et al. 2018), infectious bur sal disease (Parede et al. 2003), and avian encephalomyeli tis (Haryanto et al. 2016). An outbreak of ND can cause a devastating effect as it causes almost 100% mortality in susceptible poultry (Alexander et al. 2012). ND was also reported to have a significant economic effect on the poul try industry in Indonesia (Dharmayanti et al. 2014). New castle disease is caused by avian paramyxovirus serotipe1 (APMV1) virus, which is also known as Newcastle dis ease virus or NDV (Alexander 2003). NDV belongs to the genus Avulavirus, subfamili Paramyxovirinae, famili Paramyxoviridae, and order Mononegavirales (King et al. 2012). NDV genome is a negative sense single stranded RNA (ssRNA) encoding six viral proteins, namely nucleo capsid (N), phosphoprotein (P), matrix (M), fusion protein (F), hemaglutininneuraminidase (HN), and RNA poly merase (L) (De Leeuw andPeeters 1999; King et al. 2012). NDV infection on the host cell is mediated by two surface viral glycoproteins, the hemaglutininheuraminidase (HN) protein and fusion (F) protein (Chang and Dutch 2012). F protein in NDV plays an essential role in the viru lence of the virus (Dortmans et al. 2011). F protein can also induce protective immunity against NDV infection (Arora et al. 2010). Kim et al. (2013) reported that the F protein of NDV is a major part of protective immunity in developing genotypematched vaccine. NDV F protein consists of 553 amino acids which are synthesized as F 0 inactive precur sor (Morrison 2003). F 0 protein can be cleaved by cellular protease resulting in active F 1 and F 2 proteins. F protein is processed in trans Golgi in a mammalian cell, which produces the active form of F 1 F 2 protein with a disulfide bond (Lamb and Parks 2007). The specificity of cleavage F protein is determined by the amino acid sequence at the region of cleavage sites and varied between strain types.
In the cleavage site of F protein, low virulence NDV has one or two base amino acids. Therefore, low virulence NDV is not sensitive to intracellular protease enzyme and depends on extracellular protease in the respiratory and en teric systems of the host cells. Highly virulence NDV has multi base amino acids in the cleavage site so that it can be recognized by intracellular protease enzyme. Amino acid sequence at the cleavage site of F 0 protein is one of the fac tors that determines the systemic spreading and virulence of NDV. Highly virulence NDV contains lysine (K) and arginine (R) amino acids with 112 RRQR/KR 116 motive at C terminus F 2 and phenylalanine (F) amino acid at 117 position at N terminus protein F 1 (Ganar et al. 2014).
Newcastle disease outbreak was reported in 2009 2010 in poultry farms in Indonesia, which caused death on 7080% of the total number of chickens (Dharmayanti et al. 2014). Analysis on the amino acid identity of F and HN protein sequences from eight isolates NDV, which caused the outbreak indicated antigenic differences from La Sota and B1 vaccine strains (Xiao et al. 2012). A vac cination program has been conducted to prevent and over come ND ( [OIE] Office International of Epizootics 2012). However, ND outbreaks still continue to happen in In donesia. The outbreak is a result from antigenic differ ences between NDV strain causing the outbreak in the field with NDV strain used in the available commercial vaccine. Therefore, the vaccination program did not provide opti mal protection in the poultry post vaccination (Xiao et al. 2012; Dharmayanti et al. 2014. A recombinant vaccine is a vaccine containing anti gen of a pathogenic agent that can induce the host immu nity against the pathogenic agent (Grand et al. 2012). The recombinant vaccine is safer and more stable compared to conventional live vaccines. A recombinant vaccine is created by cloning a certain encoding gene of an immuno genic protein, expressed and purified the recombinant pro tein, and formulated it to be a vaccine (Nascimento and Leite 2012). Regardless of the type of ND vaccines in use, all vaccinated birds were fully protected from disease. All vaccinated and challenged birds show significant sero conversion after 14 days of challenge. However, some vaccinated birds were reported to shed the challenge virus from their oropharynx and cloaca, even though they are significantly lower in titers than unvaccinated challenged control birds (Jeon et al. 2008).
The development of NDV vaccines based on the phy logenetic similarity to NDV in the time of the outbreak can provide better protection against ND (Miller et al. 2007). Therefore, one of the attempts to prevent ND on poultry in Indonesia is developing an ND vaccine based on local isolates originated in Indonesia. This work aimed to ex press recombinant F protein of NDV from pBT7NHis FusionNDV expression vector which carries the F pro tein encoding gene from NDV local isolate by a cellfree protein expression system. Recombinant NDV F protein obtained from the expression is expected to be a candidate for the recombinant F vaccine to prevent ND on poultry in Indonesia.

Preparation of pBT7-N-His-Fusion-NDV recombinant plasmid
The expression vector used in this work had been prepared by Haryanto et al. (2016) and Wulanjati et al. (2018) by subcloning the F protein encoding gene of NDV from lo cal isolate in Kulon Progo, Indonesia (0663/04/2013) into pBT7NHis expression vector. The design of the recom binant plasmid vector is shown in Figure 1.

Transformation of recombinant plasmid into Escherichia coli BL21 (DE3)
Transformation of DNA plasmid pBT7NHisFusion NDV was carried out using competent Escherichia coli BL21 (DE3) strain by carefully transferring DNA plasmid into a tube containing competent cells. Plasmid DNA and competent cells were mixed gently and placed on ice for 20 min. Then, the cells were heatshocked at 42°C for 60 s. The mixture of plasmid DNA and competent cells were placed immediately on ice for 3 min. The mixture was then put into the culture medium, 10 mL of Luria Bertani (LB) (Oxoid) supplemented with 100 µg/mL ampicillin (Gold Biotechnology), and incubated at 37°C overnight. The transformation of the recombinant plasmid into E. coli generated eight growing colonies of E. coli carrying re combinant plasmid. The eight colonies of bacteria were extracted using Presto Mini Plasmid kit (Geneaid) to ob tain the recombinant plasmid DNA.

Restriction endonuclease analysis of recombinant plasmid vector by EcoRI enzyme
The screening of recombinant plasmid pBT7NHis FusionNDV was carried out by digestion of recombinant plasmid by EcoRI enzyme (Thermo Fisher Scientific) in a total volume of 10 µL. The samples were then incu bated at 37°C for 3 h. The digestion product was then visualized on a 1.5% agarose gel electrophoresis with Flu orosafe (Genetika Science Indonesia) staining. The re striction product was analyzed under UV light in the dark room.

In vitro expression of recombinant FpProtein of NDV by the cell-free protein expression system
The in vitro expression of recombinant F protein of NDV from Indonesian local isolates was performed by a cell free protein expression system using the AccuRapid pro tein expression kit (Bioneer) according to the standard manual procedure. The expressed recombinant F protein of NDV was then visualized by electrophoresis on an SDS PAGE gel followed by Coomassie Brilliant Blue R250 (Thermo Fisher Scientific) staining.

SDS-PAGE with Coomassie Brilliant Blue staining
Before SDSPAGE, the samples were denatured at 95°C for 5 min, then put into ice for about 30 min. Each sample (5 µL) was loaded on a 5% stacking gel and separated on a 12% resolving SDSPAGE gel. The protein concentration of each sample was as follow 1.39 µg/µL for sample 1, 0.13 µg/µL for sample 2, 0.88 µg/µL for sample 3, and 1.52 µg/µL for positive control. The SDSPAGE gel was run at 100 Volt for 1 h, and then it stained with Coomassie Brilliant Blue R250.

Western blotting
The expressed recombinant F protein of NDV was visu alized by Western blotting. As a positive control, Ae quore courulescens GFP fluorescent protein (AcGFP) with a molecular weight of 28.0 kDa on lane with a concentra tion of 1.52 µg/µL was used. The recombinant F protein was separated by a 12% resolving SDSPAGE gel. They were then electrotransferred onto polyvinylidene difluo ride (PVDF) membrane (Merck) for detecting the recom binant F protein. Western blotting preparation was per formed by blocking the membrane by the blocking so lution [1% BSA (Thermo Fisher Scientific) dissolved in 0.5% TweenPBS (SigmaAldrich)]. The PVDF mem brane was then incubated at room temperature for 1 h. Af ter washed three times in washing solution (0.05% Tween PBS), the PVDF membrane was probed with a mouse monoclonal antibody, anti6x histidine (his)tag antibody (Thermo Fisher Scientific) as the primary antibody in a 1:2,000 dilution. The membrane was then incubated and shaken overnight at 4°C. After three time wash ing, the PVDF membrane was subsequently incubated with the secondary antibody, alkaline phosphatase (AP) conjugated goat antimouse IgG antibody (Thermo Fisher Scientific) in a 1:15,000 dilution. The PVDF membrane was then incubated and shaken at room temperature for 1 h. After two time washing, the immunological reaction was visualized by adding NBTBCIP (Thermo Fisher Sci entific) substrate onto the PVDF membrane.

Transformation plasmid pBT-7-N-His-Fusion NDV into E. coli BL 21and visualization of recombinant NDV fusion protein expression
The transformation of pBT7NHisFusionNDV recom binant plasmid was performed initially on E. coli BL21 (DE3) competent cell. The transformation result indi cated that there were eight colonies of E. coli grew on agar LB medium (Figure 2). To confirm the result of the recombinant plasmid transformation, three from the eight colonies were selected for DNA isolation of recombinant. The next process was restriction endonuclease analysis by digestion recombinant plasmid pBT7NHisFusionNDV using Eco RI restriction enzyme. Recombinant plasmid di gestion resulted two DNA bands in size of 3,959 bp (plas mid) and 642 bp (insert gene), respectively, while the undi gested recombinant plasmid was observed to be 4,601 bp in size (Figure 3). The undigested recombinant plasmid which was run by electrophoresis in agarose gel showed three bands of DNA because it had three forms of confor mation: relaxed circular form, linearized form, and super helix form (De Mattos et al. 2004).
In Figure 4, electrophoresis using SDSPAGE of recombinant F protein on the agarose gel 12% with Coomassie Brilliant Blue staining showed that recombi nant F protein of NDV could be expressed as a thick pro FIGURE 2 Eight growing colonies of E. coli BL21 carrying recombinant plasmid pBT7-N-His-Fusion-NDV in LB agar medium with ampicillin.

FIGURE 3
The digestion result of recombinant plasmid pBT-N-His-Fusion-NDV by using Eco RI enzyme. No. 1 is recombinant plasmid of Colony-1 (C-1) was digested by Eco RI, No. 2 is an intact recombinant of C-1, No. 3 is recombinant plasmid of Colony-2 (C-2) was digested by Eco RI. No. 4 is an intact recombinant plasmid of C-2, No. 5 is recombinant plasmid of Colony-3 (C-3) was digested by Eco RI, No. 6 is an intact recombinant plasmid of C-3.
tein band with the molecular weight of 25.6 kDa (lane 1, 2, 3). Meanwhile, AcGFP with the molecular weight of 28.0 kDa on lane (+) appeared as the positive control. The recombinant F protein of NDV which expressed using cell free protein expression system found in the supernatant after incubated for 3 h at 30°C. It indicated that the ex pressed recombinant F protein of NDV in this expression system is a soluble protein. This soluble protein can be re activated and followed by a refolding process with dilution or dialysis method in refolding buffer to obtain active sol uble protein (Middelberg 2002; Burgess 2009; Yang et al. 2011.

Western blotting
Western blotting using a mouse antihistidintag antibody as the primary antibody and goat antimouse antibody as the secondary antibody was conducted to assure the result of recombinant F protein of NDV expression. The result of Western blotting can be seen in Figure 5. It showed a

Discussion
The recombinant expression plasmid that we designed pBT7NHisFusionNDV (4,601 bp) was derived from pBT7NHis plasmid which having the origin of replica tion (ORI) from pUC plasmid. This plasmid carried the F protein encoding gene from NDV which was isolated from Indonesian local isolate. Recombinant F protein expressed from pBT7NHisFusionNDV will be prepared as a can didat vaccine for viral diseases, ND, in poultry based on lo cal isolate virus. The expression of recombinant F protein from pBT7NHisFusionNDV plasmid vector was con trolled by T7 promoter (Bioneer 2016). The expression system was controlled by T7 promoter, which could be in duced through inducing lactose analog compound, namely isopropyl βDthiogalactoside (IPTG) (Rosano and Cecca relli 2014). pBT7NHisFusionNDV has a multi cloning site (MCS) and stop codon (TGA) areas with nucleotide sequences that can be digested by certain endonuclease restriction enzymes. In this plasmid, there is a ribosome binding site (RBS) that facilitates the translation process. This vector plasmid also has pUC Ori which is an ini tial area for DNA replication. In addition, there is a β lactamase encoding gene which functions in the resistance against ampicillin, which can be used in for selection of re combinant E. coli.
The expression of recombinant protein is a translation process from gene into protein through the processes of transcription and translation. Expression of protein could be performed using a prokaryotic cell system with E. coli or eukaryotic cells using yeast, filamentous fungi, and uni cellular algae. The selection of the host system depended on the target protein which was going to be expressed. In this research, the expression of recombinant NDV F pro tein was performed by using E. coli extract in a cellfree protein expression system. This system was selected be cause a prokaryotic cell is one of the selected organisms to produce a recombinant protein (Wilson and Walker 2010). In pBT7NHisFusionNDV expression plasmid, recom binant F protein is expressed as a fusion protein with 6x histag (tag polyhistidine) at the Nterminus end. After the purification process, the expressed recombinant F pro tein of NDV would be used as a candidate of recombinant vaccine against ND based on Indonesian local isolate of NDV strain. The existence of 6x histag in the recom binant F protein of NDV did not affect the structure and function of the recombinant protein (Ramos et al. 2004). Polyhistidine tag can function as the epitope for protein de tection in Western blot. Polyhistidine tag also functioned for binding with Ni 2+ in the protein purification process (Sambrook and Green 2012). The benefit of using a poly histidine tag was the recombinant F protein could be pu rified in the denatured condition because the interaction between histidine residue and NiNTA in the purification column could be stable with the existence of strong pro tein denaturant as guanidine HCl and urea. The purified recombinant protein can be renatured by slowly removing the denaturant (Terpe 2003).
In order to obtain high gene expression, the expres sion can be performed on E. coli since it grows fast, the enriched growth medium for E. coli is affordable, and the process of DNA transformation on E. coli is fast and easy (Qiagen 2001). E. coli BL21 (DE3) is a prokaryotic cell used for gene expression regulated by T7 promoter. This strain carries lysogen λDE3 containing lacI gene, RNA polymerase T7 RNA controlled by lacUV5 promoter, and a small part of lacZ gene (Wulanjati 2016). The expression of the recombinant F protein of NDV could be influenced by several factors, including vector plasmid, target protein, and strain bacteria used in the protein expression. Vari ous internal factors of the plasmid which could influence the expression are promoter, initiation region, terminator region, enhancer, ribosome binding site, origin of replica tion, resistance marker against antibiotic, and the number of plasmid copy. Meanwhile, other factors of target pro tein which could influence the expression are nucleotide sequences, amino acid sequences, secondary structures of the protein, and the suitable codon usage (Makrides 1996; Rosano andCeccarelli 2014). Besides, the productivity of the recombinant protein expression could be enhanced by optimization of external factors such as the temperature of bacterial growth and the level of expressioninducing compound (Chen et al. 2007; Volontè et al. 2008; Larentis et al. 2011. Expression of NDV F protein encoding gene have been conducted by some researchers. For example, Chen et al. (2001) conducted cloning and fulllength expres sion of cDNA from NDV F gene isolate V 4 (Queens land/66strain) on mammal's cell in Chinese Hamster Ovary (CHOK1) and its mutant line cell Lec 3.2.8.1. Park et al. (2014) developed a viruslike particles (VLP) vaccine by expressing NDV F protein with Sf9 cell to obtain recombinant protein of baculovirus (rBV). In this work, we used local isolates of NDV from Kulon Progo, Yogyakarta, Indonesia, whose pathotype characterization has been identified by Haryanto et al. (2015). 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 cellfree protein expression system is one of in vitro protein expression methods to study biological reac tions in a cell using a living cell system that reduces com plex interactions on living cells, so the transcription, trans lation, and cell metabolism processes occur in an open en vironment. This system aims to understand, utilize, and extend natural biological system capability without using living cells (Hodgman and Jewett 2012). The expressed recombinant F protein of NDV which had been purified, had the potential as a candidate of recombinant F vaccine for NDV based on the local isolate.

Conclusions
The fusion protein encoding gene of NDV subcloned into the pBT7NHis expression vector has been successfully in vitro expressed by a cellfree protein expression system. Visualization by using Coomassie Brilliant Blue staining on SDSPAGE gel and Western blot confirmed that the re combinant F protein of NDV expressed as a specific re combinant protein with a molecular weight of 25.6 kDa.