TECHNICAL FIELD

The present disclosure relates to a method of detecting Chikungunya virus in a sample by a recombinant coat peptide antigen of Chikungunya virus. The disclosure further relates to the nucleotide and peptide sequences for arriving at the recombinant coat peptide of the disclosure. Further, the method of arriving at the recombinant peptide antigen is also described herein. Further, vector and host cells obtained in the present disclosure are also described herein, along with a kit to detect the said virus.

BACKGROUND

Chikungunya is generally considered self- limiting and has been reported as non- fatal in the past. But recent reports indicated that one third of the people in the Indian Ocean island of Reunion have been affected by Chikungunya with 237 deaths. Chik virus was first reported in India in 1963 in Kolkata and it re-emerged in India in December 2005 after a gap of 32 years. It has affected a large section of the population of southern India and some parts of North India. Recently, there has been an outbreak of viral infections in Kerala, India. For antibody detection in blood serum of febrile patients, technically two types of protein antigens at present are being followed. One type is isolated from the viral culture which is a big size protein and its specificity is comparatively less. The second type is produced by recombinant methods. Recombinant protein is produced by the expression of genes of viral coat membrane and they are more specific. Today the available recombinant proteins used as antigen for detecting chik antibody are big in size, i.e, more than 48 kd. The size of the protein is proportionate to the gene size. When the protein is larger its expression and purification will be expensive and cumbersome.

The large fragments can be expressed in vector systems other than bacteria for maintaining the functional nature of the proteins. Moreover, the molecular size of recombinant protein expressed from large fragments will be high; hence the purification will be expensive. Whereas, if prokaryotic expression [bacterial expression] is possible, the production cost will be much lesser and the process will be more reliable, specific and consistent. Therefore, there is a need in the art for developing a recombinant protein which is smaller and cost effective which will overcome these constraints.

The total size of the coat protein gene is 4.3 kb (kilo base) with a protein size of more than 130 kd (kilo dalton ). Based on the huge size of the protein, any attempt of developing a small protein fragment or sub unit showing antigenic property will be a boon to the diagnostic companies for manufacturing a kit which is cost effective. In the the native protein kit, protein is comparatively larger in size isolated directly from the chik viral extract; hence it is not cost effective as well as non- specific. Another kit has recombinant protein which also has the size of more than 40kd. More over the available kit on recombinant protein is by the expression of eukaryotic system, hence its purification is expensive.

STATEMENT OF DISCLOSURE

Accordingly, the present disclosure relates to a nucleotide sequence set forth in SEQ ID No. 1; an amino acid sequence set forth in SEQ ID No. 2; a method of detecting Chikungunya virus, said method comprising acts of- a) inserting a nucleotide sequence set forth in SEQ ID No. 1 into a vector, transforming a host cell with said vector and expressing the nucleotide to obtain a recombinant peptide comprising amino acid sequence set forth in SEQ ID No. 2 and b) performing antigenicity test on sample by using the recombinant peptide to detect presence of anti- Chikungunya antibodies in the sample to detect the Chikungunya virus; a vector comprising a nucleotide sequence set forth in SEQ ID No. 1; a recombinant cell comprising vector as above; a recombinant coat peptide antigen of Chikungunya virus comprising amino acid sequence set forth in SEQ ID No. 2; a method of obtaining a recombinant coat peptide antigen of Chikungunya virus comprising amino acid sequence set forth in SEQ ID No. 2, said method comprising acts of - a) inserting a nucleotide sequence set forth in SEQ ID No. 1 into a vector, and transforming a host cell with said vector and b) expressing the nucleotide sequence to obtain recombinant coat peptide antigen of Chikungunya virus comprising amino acid sequence set forth in SEQ ID No. 2; and a Kit for detecting Chikungunya virus, said kit comprising components selected from group including nucleotide sequence set forth in SEQ ID No. 1, an amino acid sequence set forth in SEQ ID No. 2, vector comprising nucleotide sequence set forth in SEQ ID No. 1, recombinant cell comprising vector as above, recombinant coat peptide antigen of Chikungunya virus comprising amino acid sequence set forth in SEQ ID No. 2, antibodies developed against the recombinant antigen and antigenicity test reagents or any combinations thereof.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

The features of the present disclosure will become more fully apparent from the following description taken in conjunction with the accompanying drawings. Understanding that the drawings depict only several embodiments in accordance with the disclosure and are therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings:

Figure 1 illustrates a PCR amplification of the gene of. 800bp

Figure 2 illustrates an expression profile displaying the recombinant protein.

Figure 3 illustrates the specificity of the antigen for detecting the Chik virus.

Figure 4 illustrates cross reactivity of the antigen with Dengue virus.

Figure 5 illustrates purified CHIK El protein.

Figure 6 illustrates prokaryotic expression of CHIK El protein.

Figure 7 illustrates PCR CHIK El amplification,

Figure 8 illustrates detection of viral antigen using sandwich ELISA.

Figure 9 illustrates detection of antibodies using indirect ELISA.

Figure 10 illustrates A) cross reactivity with Dengue using sandwich ELISA and B) cross reactivity with Dengue using indirect ELISA.

Figure 1 1 illustrates antigenic domains.

Figure 12 illustrates major antigenic domains. DETAILED DESCRIPTION OF DISCLOSURE

The present disclosure is in relation to a nucleotide sequence set forth in SEQ ID No. 1.

The present disclosure is also in relation to an amino acid sequence set forth in SEQ ID No. 2.

The present disclosure is also in relation to a method of detecting Chikungunya virus, said method comprising acts of inserting a nucleotide sequence set forth in SEQ ID No. 1 into a vector, transforming a host cell with said vector and expressing the nucleotide to obtain a recombinant peptide comprising amino acid sequence set forth in SEQ ID No. 2 and performing antigenicity test on sample by using the recombinant peptide to detect the presence of anti-Chikungunya antibodies in the sample to detect the Chikungunya virus.

In an embodiment of the present disclosure, wherein the amino acid sequence set forth in SEQ ID No .2 is coded by corresponding nucleotide sequence set forth in SEQ ID No. 1, and wherein the amino acid sequence codes for an El coat peptide antigen of Chikungunya virus.

The present disclosure is also in relation to a vector comprising a nucleotide sequence set forth in SEQ ID No. 1.

The present disclosure relates to a recombinant cell comprising vector as above.

In an embodiment of the present disclosure the vector is selected from group comprising pTZ57R/T, pET32c and pET32b; and the cell is selected from group comprising JM109 and BL21DE3pLys.

The present disclosure is also in relation to a recombinant coat peptide antigen of Chikungunya virus comprising amino acid sequence set forth in SEQ ID No. 2. In still another embodiment of the present disclosure, wherein the recombinant coat peptide antigen is a recombinant El coat peptide antigen and is about 26kD in size.

The present disclosure is also in relation to a method of obtaining a recombinant coat peptide antigen of Chikungunya virus comprising amino acid sequence set forth in SEQ ID No. 2, said method comprising acts of inserting a nucleotide sequence set forth in SEQ ID No. 1 into a vector, and transforming a host cell with said vector and expressing the nucleotide sequence to obtain a recombinant coat peptide antigen of Chikungunya virus comprising amino acid sequence set forth in SEQ ID No. 2.

The present disclosure is also in relation to a kit for detecting Chikungunya virus, said kit comprising components selected from group including nucleotide sequence set forth in SEQ ID No. 1 ; an amino acid sequence set forth in SEQ ID No. 2; vector comprising nucleotide sequence set forth in SEQ ID No. 1; recombinant cell comprising vector as above; recombinant coat peptide antigen of Chikungunya virus comprising amino acid sequence set forth in SEQ ID No. 2; antibodies developed against the recombinant antigen; and antigenicity test reagents or any combinations thereof.

In an embodiment of the present disclosure, wherein the antigenicity test is ELISA.

In still another embodiment of the present disclosure the SEQ ID No.l is nucleic acid sequence for CHIK El coat protein gene of Chikungunya virus which contains 800 base pairs. In the present disclosure a genomic analysis of the gene sequence is being done and the region with more than 10 antigenic domains with two major regions in gene fragment of 800bp located at the position of 2570- 3370 of El gene; is identified. Figures 11 and 12 relate to antigenic domains and major antigenic domains. In still another embodiment of the present disclosure, the SED ID No. 2 is an amino acid sequence for CHIK El coat protein of Chikungunya virus which contains 266 amino acid residues. In an embodiment of the present disclosure, a new recombinant El coat peptide of 26 Kd size with antigenic property for the early diagnosis of Chik virus is developed. Technically, the diagnostic protocol is called as the Indirect ELISA of IgM antibody detection in the serum of febrile patients using new recombinant El coat protein antigen of Chik virus. This is the first time a small recombinant coat protein of size 26 Kd of Chik virus having antigenic property has been produced. Moreover, the recombinant protein that is expressed is from a gene fragment of size 800 bp unlike the earlier reports where either the total viral protein as such from the viral culture or recombinant protein from large gene fragments more than 1000 bp was developed. Figure 9 illustrates detection of antibodies using indirect ELISA.

In an embodiment of the present disclosure, since the size of the protein produced by a prokaryotic system (bacterial vector) is 26kd, the production cost is less compared to eukaryotic system. Since the protein size is small, it is more reliable, specific and consistent in showing the antigenic property. Prokaryotic expression of Chik El recombinant protein for ELISA to detect Chik viral antibodies in patient serum is reported. Production of recombinant protein by prokaryotic expression in a biologically active form that is used in ELISA experiment is also presented. Since only a portion of the coat protein gene is used for recombinant protein production, usage of whole virus or viral cultures for preparing antigen is avoided and thereby reducing the risk associated.

Since the strain variation reported in the phylogenetic analysis chik viruses is purely based on single nucleotide polymorphisms (SNPs), these types of variations no where affect the antigenic property of the El structural protein as such. So in the preparation diagnostic kits, it has not much relevance. So the present recombinant protein is used to detect chik viral antibodies irrespective of the geographical variations noted in the viral stains. In an embodiment of the present disclosure, the kit based on the present recombinant protein is used to detect all strains of chik viral antibodies irrespective of the strain variations. Further, techniques used in the prior art for detection are RT-PCR based technique and ELISA based technique. RT-PCR based detection is costly and time consuming technique and the chance for contamination and false positive is high. However, IgM capturing ELISA based technique using the instant recombinant protein is cheaper, less time consuming for the early detection of viral antibodies. The recombinant protein produced is of a subunit protein (since only a fragment of 800bp the El coat protein gene is used not the entire gene). The recombinant protein produced from the partial gene itself has antigenic domains specific to chik viral antibodies.

The present disclosure also relates to the production of recombinant antigen of CHIK virus in prokaryotic system. In the prior art, the 40kDa protein that produced was a recombinant one in eukaryotic vector. This change reflects the cost difference noticed during the purification of protein. Purification of the recombinant protein from a eukaryotic system is expensive and cumbersome. The objective of the present finding is to develop a recombinant protein of the Chik viral coat gene with antigenic property for developing a native molecular diagnostic kit for Chik viral detection. Proper clinical management of the Chikungunya disease remains as a constraint due to the lack of specific diagnostic methods. Since the phenotypic symptoms of both dengue and Chik viruses showed close similarities the differential diagnosis of these infections is essential for effective clinical management and epidemiological studies.

The present disclosure relates to:

a) Using the recombinant antigen of 26kDa developed against the polyclonal antibody against CHIK antigen and purifying the same; and developing the sandwich ELISA for detecting the antigen in CHIK viral patients. The antibody produced from recombinant antigen is treated as primary antibody for sandwich ELISA.

b) Isolating Human anti-CHIK IgG antibody from acute CHIK patients. This antibody is used as capture antibody.

c) Using the primary and capture antibody, a protocol for sandwich ELISA has been standardized and developed for detection of viral antigens of serum of patients.

d) More than 100 samples are used for the study. Sensitivity, specificity and cross reactivity of the test is confirmed. The data is presented in the form of figures of sandwich ELISA test. Figure 8 illustrates detection of viral antigen using sandwich ELISA.

In still another embodiment of the present invention, wherein the reagents used for Antigenicity tests are selected from a group comprising Coating, Blocking buffer, Wash Buffer, Sample diluents, Conjugate Diluent, CHIK positive control, CHIK Negative Control, Substrate, Secondary antibody and Stop Solution.

In still another embodiment of the present invention, wherein the Coating of the ELISA strips with renatured and filter sterilized recombinant coat peptide is done at 20 micro gram per well, overnight at 4 °C; blocking buffer is 5% BSA dissolved in Phosphate buffer Saline; wash buffer is Phosphate buffer saline with 0.05% triton; sample diluents are Phosphate buffer saline-9%, BSA -2% and Sodium azide-0.01%; conjugate diluents is Sodium Chloride- 0.25M, Sodium Phosphate- 0.01M and BSA - 15 mg/ml at pH 7.6; CHIK positive control is Inactivated anti chik positive human serum-O.lml and Sodium azide- 0.01%; CHIK negative control is Normal human serum-O.lml and Sodium azide- 0.01%; substrate is Substrate -TMB ( 3,3',5,5' -Tetra methyl Benzidine); Secondary antibody are HRP conjugated goat anti human IgM antibody and the Stop Solution is IN H ₂ S0 ₄ The present disclosure relates to an ELISA kit using recombinant antigen of 26kDa is developed for detecting the CHIK antibodies in the serum samples of patients. The test is found rapid, specific, and cost effective. Subsequently, study is carried out for developing polyclonal antibodies of the instant recombinant antigen in rabbit. The study is to develop sandwich ELISA kit for detecting the CHIK antigen in the serum samples of patients. The advantage of the sandwich ELISA diagnostics is that one can detect the disease at the earlier phase itself that is the first day of fever unlike the antigen based ELISA. Hence, the present relates to:

· Antigen based ELISA kit for detecting antibody.

• Antigen capture sandwich ELISA for detecting viral antigen in serum samples of patients.

• Diagnostic kit with recombinant antigen for detecting the antibodies of serum samples.

· Diagnostic kit with antibodies (developed from recombinant antigen) for detecting the antigen of serum samples.

The following examples provide further illustrations and embodiments of the present disclosure. However, the examples should not be construed to limit the scope of the present disclosure.

EXAMPLES

Example 1: cDNA synthesis of viral RNA

The presence of viral RNA in the Chikungunya virus was identified, employing Chik specific primers of structural El gene. The primers from the viral coat genome for a gene product of size 800bp were designed. Total RNA was extracted from Chikungunya infected patient serum using GF -Viral Nucleic acid Extraction Kit (Vivantis) according to the manufacturer's instructions. The extracted RNA was eluted in 30μ1 of elution buffer and then used as a template for reverse transcription PCR (RT-PCR) following the method of AY Joseph et al, 2008.The cDNA was subjected to PCR assay and an amplicon of expected size ie. 800bp was obtained. Figure 1 illustrates a PCR amplification of the gene of. 800bp.

Example 2: Sequencing Analysis

The amplified cDNA was cloned and sequenced and the nucleotide sequence of El coat gene showed 100% homology with Chik viral coat genome sequence of genbank.

Example 3: Protein Expression and purification

The purified PCR product of size 800bp was used for ligation in cloning vector and transformed. The genes were digested and directly cloned into the prokaryotic expression vector pET32b (Novagen), which contains a his-tag on the C terminus. The ligated products were transformed into E.coli BL 21 DE3PLys cells and the transformants were selected on LB agar plate in presence of ampicillin (50μg/ml). The transformed E.coli BL 21 cells were grown at 37°C to an OD ₆ oo of 0.5-0.6 in LB medium containing ampicillin (50μg/ml). Recombinant clones expression were induced by the addition of 0.1M IPTG for 3hr with shaking. Aliquots of the induced and un-induced cultures were analysed by SDS-PAGE prior to the purification. The induced cultures were centrifuged at 12,000 rpm for 5 minutes at 4°C.The cell pellet was suspended in IX Phosphate Buffered Saline (pH 8) to a homogenous suspension and centrifuged again to obtain pellet. The cell pellets were re-suspended in same buffer supplemented with Lysozyme (lOmg/ml) and aprotenin (lmg/ml). After being frozen and thawed ten times, cell lysate treated with DNase (lmg/ml) and RNase (lmg/ml) and incubated for 10 min at 4°C. The resulting cell lysates were centrifuged at 10,000 rpm for 30minutes at 4°C. The cell pellets were solubilized in 8M urea and centrifuged at 14,000 rpm for lhr to remove any insoluble debris. The recombinants expressed from both these strains are purified based on its C- terminal His-tag by Ni-affinity column chromatography. The concentration of the protein was determined by Bradford method and the purity and the size of the protein were analyzed by Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis (SDS- PAGE). The size of the fusion protein was noticed as 47kd in the gel, in which 26 kd is the size of the recombinant protein and the remaining is the his- tag i.e. 21kd. Figure 2 illustrates an expression profile displaying the recombinant protein. Figure 6 illustrates prokaryotic expression of CHIK El protein and Figure 7 illustrates PCR CHIK El amplification,

Example 4: Production of recombinant antigen in bacterial system

The gene is subcloned into prokaryotic expression vector pET32c and transformed with BL21DE3pLys cells. Protein expression is induced by adding IPTG at a final concentration of ImM for 3 h. 1 ml of each samples is collected at regular intervals starting from 0 h to 3 h. The expression profile is determined by SDS-PAGE analysis. For purifying the recombinant CHIK El protein, the 50 ml culture is harvested after 3 h of induction. The culture is centrifuged at 5000 g for 5 min at 4 °C and the pellet is resuspended in 1 ml ice-cold phosphate buffered saline (PBS) pH 7.4. The cell suspension is centrifuged and the PBS ish is repeated twice. The cell pellet is finally resuspended in 1 ml PBS and to it 5 μΐ aprotinin (1 mg/ml) and 10 μΐ of lysozyme (10 mg/ml) were added. Cell lysis is done by repeated freeze -thawing in liquid nitrogen (20 s) and at 37 °C (1 min). 10 μΐ of DNase (1 mg/ml) and R ase A (10 mg/ml) is added to the lysate and incubated at 4 °C for 10 min with shaking. After the incubation the lysate is centrifuged at 12,000 g for 30 min at 4 °C. The supernatant is collected and the inclusion body pellet is resuspended in buffer A containing 200mM NaH2P04, 500mM NaCl, 5mM Imidazole and 8M urea and incubated at 4 °C for 1 h. The solution is centrifuged at 14,000 g for 60 min and the supernatant collected and stored as the source of recombinant CHIK El protein. The recombinant protein is purified by affinity chromatography using Ni2+ coated IMAC Hypercel column (PALL) using elution buffer containing 200mM Imidazole. The eluted protein is dialyzed against PBS containing decreasing concentrations of urea (6M to 0 M). Figure 5 relates to purified CHIK El protein.

Example 5: Antigenicity test by ELISA

Ninety-six well micro titer plates were coated with serial dilutions of recombinant Chik protein in different concentrations (0.25, 0.5, 1, 2.5, 5, 10, 15 and 20μg) in PBS (pH7.4) overnight at 4°C. Plates were washed and blocked with 5% BSA overnight at 4°C. The positive serum samples of Chikungunya patients and negative control samples collected from the approved diagnostic centers and labs were diluted in PBS containing 2% BSA (1 : 100) and added to the wells as primary antibody. For cross reactivity test, positive samples of dengue patients were taken. The plates were incubated for 30 minutes. After the incubation the plates were washed thoroughly for removing the unbound antibodies using PBS containing 0.5% Tween 20. Horse radish peroxidase (HRP) conjugated Goat anti-human IgM antibody (1 :5000 dilution,) was added to the wells as secondary antibody. The plates were washed thoroughly four times with PBS containing 0.05% Tween20. TMB (3,3',5.5'-Tetramethynebenzidine)/ H2O2, the substrate of the enzyme peroxidase was added to the wells and incubated for 10 min at 37°C. A blue colour complex appeared indicating peroxidase - TMB reaction. The reaction was stopped by adding IN H2SO4 and the absorbance was read at 450nm using an automatic ELISA plate reader. Figure 10 illustrates A) cross reactivity with Dengue using sandwich ELISA and B) cross reactivity with Dengue using indirect ELISA. Results:

All the positive samples of the patients used for the study showed positive reading in OD value indicating the reaction of the recombinant antigen on the primary antibody. The negative samples exhibit their limitation as negative. Figure 3 illustrates the specificity of the antigen for detecting the Chik virus.

Thus the antigenic property of the recombinant protein was confirmed. The specificity of the antigen exclusively on Chik antibody was ascertained by the negative response during cross reactivity test of the positive samples of Dengue patients. Figure 4 illustrates cross reactivity of the antigen with Dengue virus. Even though the colour indication of the antigen- antibody interaction appeared from 5 μg antigen concentration onwards, 20μg was selected as optimum concentration of the antigen. SEQUENCE LISTING

<110> SCMS, INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY REASEARCH AND DEVELOPMENT

<120> NUCLEOTIDE AND AMINO ACID SEQUENCE, METHODS AND KIT THEREOF

<130> IP15710

<140> 980/CHE/201 1

<141> 2011-03-28

<160> 2

<170> Patentln version 3.5

<210> 1

<21 1> 800

<212> DNA

<213> Chikungunya viras

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Met Val Leu Glu Met Glu Leu Leu Ser Val Thr Leu Glu Pro Thr Leu

1 5 10 15

Ser Leu Asp Tyr He Thr Cys Glu Tyr Lys Thr Val He Pro Ser Pro

20 25 30

Tyr Val Lys Cys Cys Gly Thr Ala Glu Cys Lys Asp Lys Asn Leu Pro

35 40 45

Asp Tyr Ser Cys Lys Val Phe Thr Gly Val Tyr Pro Phe Met Trp Gly

50 55 60

Gly Ala Tyr Cys Phe Cys Asp Ala Glu Asn Thr Gin Leu Ser Glu Ala

65 70 75 80 His Val Glu Lys Ser Glu Ser Cys Lys Thr Glu Phe Ala Ser Ala Tyr

85 90 95

Arg Ala His Thr Ala Ser Ala Ser Ala Lys Leu Arg Val Leu Tyr Gin 100 105 1 10

Gly Asn Asn He Thr Val Thr Ala Tyr Ala Asn Gly Asp His Ala Val 115 120 125

Thr Val Lys Asp Ala Lys Phe He Val Gly Pro Met Ser Ser Ala Trp

130 135 140

Thr Pro Phe Asp Asn Lys He Val Val Tyr Lys Gly Asp Val Tyr Asn 145 150 155 160

Met Asp Tyr Pro Pro Phe Gly Ala Gly Arg Pro Gly Gin Phe Gly Asp

165 170 175

He Gin Ser Arg Thr Pro Glu Ser Lys Asp Val Tyr Ala Asn Thr Gin 180 185 190

Leu Val Leu Gin Arg Pro Ala Ala Gly Thr Val His Val Pro Tyr Ser 195 200 205

Gin Ala Pro Ser Gly Phe Lys Tyr Trp Leu Lys Glu Arg Gly Ala Ser

210 215 220

Leu Gin His Thr Ala Pro Phe Gly Cys Gin He Ala Thr Asn Pro Val 225 230 235 240

Arg Ala Val Asn Cys Ala Val Gly Asn Met Pro He Ser He Asp He

245 250 255

Pro Glu Ala Ala Phe Thr Arg Val Val Asp

260 265