THEREOF"

TECHNICAL FIELD

The present disclosure belongs to the field of molecular biology. The present disclosure relates to the nucleotide and amino acid sequences coding for Lactoferrin (Lf) gene of Vechur breed of Bos indicus comprising 9 Single Nucleotide Polymorphisms (SNP's) and 7 amino acid variations corresponding to the 9 SNP's respectively. The present disclosure further relates to vector and host cell comprising said sequences and obtaining recombinant peptide thereof. The present disclosure also relates to transgenic products comprising the nucleotide sequence and process for obtaining the transgenic product.

BACKGROUND OF THE DISCLOSURE

Many full length Lactoferrin coding sequences have been reported from the species Bos.taurus (exotic) and the hybrid of Bos.taurus & Bos indicus, but none so far from Bos indicus (Native).

Lactoferrin is an iron-binding glycoprotein found in the milk and other exocrine secretions of mammalian species. The Vechur breed of Bos indicus is unique due to its extremely small size, easy adaptability and resistance to diseases. It is approved as the smallest cow as per the Guinness world record.

The Lactoferrin protein continues to be very popular among researchers worldwide owing to its varied nutritional and therapeutic properties as evident from numerous studies. Despite of this not much has been done to characterize lactoferrin in the numerous cattle breeds found in India, with its rare indigenous breeds not an exception. Among the indigenous cattle found in India, Vechur breed from Kerala is remarkable due to the smallest size, low fodder intake and good adaptability. This breed is also not an exception as far as studies on its Lactoferrin are concerned. The Lf gene has not been isolated or sequenced from Vechur till now. STATEMENT OF THE DISCLOSURE

Accordingly, the present disclosure relates to nucleotide sequence set forth as SEQ ID Nos. 1 or 3 or nucleotide sequence comprising sequence set forth as SEQ ID Nos.l or 3; amino acid sequence set forth as SEQ ID Nos. 2 or 5 or amino acid sequence comprising sequence set forth as SEQ ID Nos. 2 or 5; a vector comprising nucleotide sequences as above; a transformed host cell comprising vector as above; a recombinant peptide comprising amino acid sequences as above; a process of isolating recombinant peptide, said process comprising acts of- a) inserting nucleotide sequence set forth as SEQ ID Nos. 1 or 3, or nucleotide sequence comprising sequence set forth as SEQ ID Nos.l or 3 into a vector, and transforming a host cell with said vector and b) expressing the nucleotide sequence to obtain the recombinant protein peptide as above; a transgenic product comprising nucleotide sequence as above; a process of obtaining transgenic product, said process comprising acts of - a)isolating nucleotide sequence set forth as SEQ ID Nos. 1 or 3, or nucleotide sequence comprising sequence set forth as SEQ ID Nos.l or 3 and inserting the sequence into genome of a wild type cell and b) expressing the nucleotide sequence as recombinant peptide to obtain the transgenic product; and single nucleotide polymorphism (SNP) in a nucleotide sequence set forth as SEQ ID No. 1 or 3, or nucleotide sequence comprising sequence set forth as SEQ ID No.l or 3, at positions selected from group comprising 187, 531, 795, 984, 1287, 1791, 1874, 1884 and 1900 or any combination thereof.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein, wherein;

Figure 1 illustrates a PCR amplification of the Lf gene of 2.14Kb from Vechur cow's Milk.

Figure 2 illustrates the Nucleotide sequence of the Lf gene from Vechur breed of cow with marked SNPs. Figure 3 illustrates nucleotide sequence alignment of Lf gene from Vechur breed of Bos indicus [BLfV] and Bos taurus [L08604].

Figure 4 illustrates an amino acid sequence of Vechur Lactoferrin (Lf).

Figure 5 illustrates amino acid sequence alignment of Lf gene from Vechur breed of Bos indicus [BLf V3 aa] and Bos taurus [BLf Bt aa]

Figure 6 illustrates tertiary structure of Lf protein from Bos taurus.

Figure 7 illustrates tertiary structure of Lf protein from Vechur breed of Bos indicus. Figure 8 illustrates tertiary structure of Lf protein from Vechur breed of Bos indicus. Figure 9 illustrates BLfV tertiary structure showing Arginine at position 176 [green colour].

Figure 10 illustrates BLfV tertiary structure showing Arginine at position 264 [yellow colour].

Figure 11 illustrates BLfV tertiary structure showing Arginine at position 632 [orange colour].

Figure 12 illustrates Antimicrobial Sensitivity Test Data of recombinant lactoferrin.

Figure 13 illustrates PCR amplification of Vechur Lactoferrin gene from peanut DNA. Figure 14 illustrates PCR amplification of Vechur Lactoferrin gene from Banana DNA. Figure 15 illustrates comparison of antibacterial activity of Bos Indicus [Vechur] and

Bos Taurus.

Figure 16 illustrates antibacterial activity of recombinant protein of Bos Indicus Vechur breed compared with Bos Taurus.

Figure 17 illustrates antimicrobial activity of Vechur (hydrolysate).

Figure 18 illustrates antimicrobial activity of Vechur (Rec.BLf).

Figure 19 illustrates antimicrobial activity of Vechur (hydrolysate + Amp.).

Figure 20 illustrates N terminal region of lactoferrin gene ( 300bp ) with Lactoferricin domain. Nucleotide sequence of Lactoferrin (300bp) with lactoferricin region as SEQ ID No. 3 and amino acid sequence corresponding to Lactoferricin as SEQ ID No. 5.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to Nucleotide sequence set forth as SEQ ID Nos. 1 or 3 or nucleotide sequence comprising sequence set forth as SEQ ID Nos.l or 3. The present disclosure relates to amino acid sequence set forth as SEQ ID Nos. 2 or 5 or amino acid sequence comprising sequence set forth as SEQ ID Nos. 2 or 5.

In an embodiment of the present disclosure, the nucleotide sequence comprises 9 single nucleotide polymorphisms (SNPs) and codes for Lactoferrin gene of Vechur breed of Bos indicus; and wherein the amino acid sequence comprises 7 amino acid variations corresponding to the 9 SNPs.

The present disclosure relates to a vector comprising nucleotide sequences as above. The present disclosure relates to a transformed host cell comprising vector as above. The present disclosure relates to a recombinant peptide comprising amino acid sequences as above.

The present disclosure relates to a process of isolating recombinant peptide, said process comprising acts of:

a) inserting nucleotide sequence set forth as SEQ ID Nos. 1 or 3, or nucleotide sequence comprising sequence set forth as SEQ ID Nos.l or 3 into a vector, and transforming a host cell with said vector; and b) expressing the nucleotide sequence to obtain the recombinant protein peptide as above.

The present disclosure relates to a transgenic product comprising nucleotide sequence as above.

In an embodiment of the present disclosure, the transgenic product is selected from group comprising milk, peanut and banana.

In another embodiment of the present disclosure, the vector is selected from group comprising Plasmid, TA cloning vector, pTZ57R/T, pOREE2, pTZ57R/T

The present disclosure to relates a process of obtaining transgenic product, said process comprising acts of:

a) isolating nucleotide sequence set forth as SEQ ID Nos. 1 or 3, or nucleotide sequence comprising sequence set forth as SEQ ID Nos.l or 3 and inserting the sequence into genome of a wild type cell; and b) expressing the nucleotide sequence as recombinant peptide to obtain the transgenic product. The present disclosure relates single nucleotide polymorphism (SNP) in a nucleotide sequence set forth as SEQ ID No. 1 or 3, or nucleotide sequence comprising sequence set forth as SEQ ID No.l or 3, at positions selected from group comprising 187, 531 , 795, 984, 1287, 1791 , 1874, 1884 and 1900 or any combination thereof.

The present disclosure relates to a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l (Figures 1&2).

The present disclosure also relates to an amino acid sequence set forth as SEQ ID No. 2 or an amino acid sequence comprising sequence set forth as SEQ ID No. 2 (Figure 4).

In an embodiment of the present disclosure, the nucleotide sequence comprises 9 single nucleotide polymorphisms (SNPs) and codes for Lactoferrin gene of Vechur breed of Bos indicus; and wherein the amino acid sequence comprises 7 amino acid variations corresponding to the 9 SNPs (Figures 3 & 5).

The present disclosure also relates to a process of isolating a recombinant peptide, said process comprising acts of:

a) inserting a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l into a vector, and transforming a host cell with said vector; and

b) expressing the nucleotide sequence to obtain a recombinant protein peptide as above (Figures 6,7 &8).

In an embodiment of the present disclosure, the transgenic product is selected from a group comprising milk, peanut and banana (Figures 13 & 14).

The present disclosure also relates to single nucleotide polymorphism (SNP) in a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l, at positions selected from a group comprising 187, 531 , 795, 984, 1287, 1791, 1874, 1884 and 1900 or any combination thereof (Figure 3). The present disclosure relates to a full length lactoferrin coding region from the critical breed Vechur variety of Bos indicus. The Lf gene has not been isolated or sequenced from Vechur in the past. However, the present disclosure relates to the gene being amplified and sequenced from the Vechur breed of cattle.

The present disclosure represents the sequence of the full coding region of the Lf gene. Considering the multidimensional therapeutic protein, this disclosure has a lot of commercial relevance in the pharmaceutical industry. The present disclosure discloses that the full length coding region of the lactoferrin gene has been cloned and sequenced in Vechur, the critical breed (Figures 1&2). The sequence analysis of lactoferrin gene of Vechur cow (BLfV) showed nine Single nucleotide polymorphisms (SNPs) within the exon sequence of the gene at positions viz. 187(C/T), 531(T/C), 795 (TIC), 984(T/C), 1287 (C/T), 1791(A/T), 1874(A/C), 1884(A/G) & 1900 (A/G) in which the variations at 187, 531, 795, 1287, 1791 , 1884 and 1900 positions were associated with an amino acid substitutions from Alanine to Valine at 61 position, Cysteine to Arginine at 176 position, Cysteine to Arginine at 264 position, Proline to Serine at 428 position,Threonine to Serine at 596 position, Lysine to Glutamic acid at 627 position and Histidine to Arginine at 632 position of the protein sequence respectively( Figure 3 & 5). The amino acid change from cysteine to arginine that was obtained in lactoferrin protein of Vechur cow remains as a unique variation compared to the lactoferrin of all the breeds of cows examined (Figures 9, 10 &11).

In an embodiment of the present disclosure, prior research work has not been done so far on lactoferrin (Lf) gene of Bos indicus vechur breed at molecular level. The present disclosure states that the lactoferrin gene of vechur has been cloned, sequenced and characterized at national and international level.

Moreover, the Lactoferrin is a glycoprotein predominant in the milk of all mammals for the protection and growth of infants from all diseases. Besides its innate function of protection it has multidimensional functions viz. antibacterial, antifungal, antiviral, anti inflammatory, and immuno stimulant. The gene for lactoferrin is distributed in 17 exons of DNA with a size of at 35 kb. The coding region of the gene is at 2.15 kb (Figure 1). The variation of 9 SNPS and 7 amino acid substitutions are unique and exclusive to Lf gene of vechur breed (Figures 3&5).

The present disclosure also highlights the pronounced substitution of amino acid arginine in the study because irrespective all the other amino acid changes high arginine level emphasizes the therapeutic property as an immunostimulant for inducing resistance. The positions of the amino acid arginine in the tertiary structure clearly support its functional role in the protein. From the structure it can be derived that the arginine change is in the helical and beta chain of the protein. For the sequence comparison, the so far available sequences of B. taurus has been considered since B. taurus and B. indicus are pure line breeds (Figures 9, 10 &11). RNA Isolation and Amplification of the gene:

Fresh milk samples were collected in sterilized bottles from Vechur cows at College of Veterinary and Animal Sciences, Mannuthy and brought to the lab in ice. RNA was isolated from the milk samples by following the protocol of Talebkhan Garoussi et al (2007) with slight modifications. The RNA was converted to cDNA by using Poly-T primer and AMV-RT enzyme. The cDNA was used to amplify the gene by PCR using Gene specific primers designed as disclosed in the present disclosure. The amplified gene was visible as a band of 2.141 kb size on agarose gel. The gene was eluted and ligated into a TA cloning vector. The ligation mix was transformed and the plasmid was isolated from positive recombinants and sequenced. The sequence was compared and analysed with the sequence of Bos. taurus and the hybrids of B. taurus & B. indicus for detecting the Single nucleotide polymorphism (SNP) and amino acid variation.

In all the lactoferrin gene study so far done in cows, RNA isolations were made from tissues and blood samples. In the present disclosure, milk samples were used for isolating RNA. Since it is a critical breed, milk is preferred for isolating RNA rather than blood and tissues. As a vulnerable breed of Bos indicus, the present disclosure intends to conserve the lactoferrin gene of vechur cow by transforming the gene to a suitable host for maintaining its functionality, through suitable promoters. Subsequent to the prokaryotic expression of lactoferrin protein, the gene was successfully transformed to the edible crops peanut and banana, with an objective of exploiting the therapeutic properties of vechur lactoferrin protein, including antimicrobial activity of the products. Therefore, this is the first time a transgenic crop has been developed with lactoferrin gene of vechur (Figures 13 & 14). The graph in Figure 12 demonstrates the antimicrobial activity of vechur recombinant lactoferrin compared with native lactoferrin. The results of antimicrobial sensitivity test data is depicted in table 1 below:

Table 1 : Antimicrobial sensitivity test data

[Organism used: Enteropathogenic (EPEC) E. coli (0111)]

The higher efficacy of vechur lactoferrin inhibiting the growth of microbes is obvious from the figure.

Despite the identity noticed in the nucleotide sequence of the lactoferrin genes of cows, the amino acid substitutions in the sequence as well as their positions in the sequences have a pertinent role in determining the functionality of a gene. For eg. GADPH genes in human system are some what identical in all the human species. At the same time the genes exhibit the property of pseudogenes at 62% level, due to sequence variation in human population. In other words nucleotide variants and amino acid substitutions are not mere variations; they have a decisive role in determining the nature of the gene. In the case of lactoferrin gene of vechur cow, it stands as an invention due to its structure as well as its function from other lactoferrin genes. So the amino acid substitutions of the present disclosure remains as the crucial factor in regulating the therapeutic functions of the vechur cow's milk.

The sequences disclosed in the present disclosure involve 9 SNPs with 7 amino acid substitutions. In this arginine substitutions are at three positions: 176, 264 and 632 (Figures 9, 10 &1 1). The presence of the amino acid arginine in the milk is the factor for enhancing the therapeutic property. Any variation in the amino acids present in the functional region of the protein sequences is relevant in deciding the property of the protein in one way or other.

As a breed localized in a geographical pocket of Kerala, the possibility of gene variation must be less compared to other breeds. Hence the variations are unique. Vechur cow still maintains a wild nature due to its isolation from other germplasm. SNPs leading to significant amino acid changes only are important. The properties of a protein depend upon its folding and 3 D structure which in turn depends upon the amino acid seq. Certain changes in amino acids bring about changes in the total structure which may cause changes in properties. The present disclosure is further elaborated by way of the following examples and accompanying figures herein. However, the elaborations and illustrations herein should not be construed to limit the scope of the present disclosure.

EXAMPLES

Example 1:

In the full coding region of 2.12 kb of Lf, at the N-terminal region of the gene of a size of 300bp exhibits the presence of the powerful antibacterial domain is the specific reason for antimicrobial potential of Lf of Vechur cow.

The effect of recombinant lactoferrin in inhibiting the bacterial growth is demonstrated in figure 16. The data indicates active suppression of bacterial growth by bovine Lactoferrin of vechur cow (Bos indicus), which is significantly higher than the recombinant lactoferrin protein of Bos taurus hybrid (Figures 16).

The Bovine lactoferrin (300bp) sequence SEQ ID No. 3 (Figure 20) was obtained by amplification process of the gene disclosed as SEQ ID No. 1 using the following primers:

SEQ ID No. 6: Forward primer:

5'-ggatccccagccatgaagctc- 3'

SEQ ID No. 7: Reverse primer :

5 ' -tgctgctactggccgcagtt-3 '

The conditions required for the amplification process are:

1. The concentration of ampicillin in all the standards and the experiment is 20μg (50mg/ml is the stock)

2. Amplification condition of 300bp lactoferrin gene of Vechur

Initial denaturation - 94°C

Denaturation - 94°C

Annealing - 55 °C

Extension - 72°C

Final extension - 72°C

Hold - 4°C

Example 2:

Antibacterial sensitivity test are carried out under three phases for determining the antibacterial property of recombinant lactoferrin of vechur cow.

Phase 1: Bacterial culture as such (Control); bacterial culture with ampicillin as

(Standard); bacterial culture with undigested recombinant protein of vechur cow. Phase 2: Control; standard; bacterial culture with pepsin digested recombinant lactoferrin of vechur cow.

Phase 3: Control; standard; bacterial culture with pepsin digested recombinant lactoferrin along with along with ampicillin in the ratio 50(^g:20 μg. The figures 17, 18 and 19 show the results of phase 1 , 2 and 3, respectively.

From the results obtained from phase 1 and phase 2 experiments, one infers that the recombinant protein in both forms, digested and undigested, exhibit suppression of bacterial growth similar to that of the standard. But in case of phase 3 a significant level of suppression of bacterial growth is noticed, with additive effect of ampicillin along with the digested recombinant lactoferrin even though the concentration of the ampicillin used is negligible (20 μg). So this synergistic effect of digested recombinant protein and ampicillin in bacterial culture medium can be interpreted in 3 ways:

i) Being an iron binding glycoprotein lactoferrin exhibit its capability in absorbing iron from the culture medium.

ii) Based on the therapeutic property of lactoferrin protein, it disintegrates the bacterial cell membrane that indirectly disturbs the cell transport.

iii) Coinciding with the usual property of ampicillin its presence in the bacterial medium along with the digested recombinant lactoferrin activates the membrane disintegration as an additive.

Protocol for obtaining Transgenic Peanut & Banana producing Lactoferrin Protein of Vechur cow

Example 3:

Production of Transgenic Peanut:

R A was isolated from milk of Vechur cow. Lactoferrin (Lf) gene was amplified from the milk RNA by RT-PCR using specifically designed primers. Lf gene was ligated into pTZ57R/T vector and sub cloned into pOREE2 vector after releasing from pTZ57R/T vector using Sacl and Sail restriction enzymes.

The recombinant pOREE2 plasmid containing Lf gene (BLf-pOREE2) was used to transform Agrobacterium tumefaciens strain EHA 105. Peanut embryos were developed for cocultivation with the EHA105 strain containing BLf-pOREE2 plasmid.

The peanut embryos were incubated with A. tumefaciens cell suspension containing BLf- pOREE2 plasmid. Subsequently peanut embryos with A. tumefaciens were incubated on solid medium in dark for three days.

Later the peanut embryos were transferred to suitable medium containing cefotaxime for regeneration. The embryos were sub-cultured in medium containing the marker antibiotic, Kanamycin. The transformants were selected for screening i.e. DNA isolation from peanut leaves followed by PCR using BLf primers.

The transformed peanut plants after rooting and hardening were transferred to soil.

Example 4:

Production of Transgenic Banana:

RNA was isolated from milk of Vechur cow. Lactoferrin (Lf) gene was amplified from the milk RNA by RT-PCR using designed primers. Lf gene was ligated into pTZ57R/T vector and sub cloned into pOREE2 vector after releasing from pTZ57R/T vector using Sacl and Sail restriction enzymes.

The recombinant pOREE2 plasmid containing Lf gene (BLf-pOREE2) was used to transform Agrobacterium tumefaciens strain EHA 105. The banana corms were processed and cultured in shooting medium. The tissue cultured banana corms were used for cocultivation with the EHA105 strain containing BLf-pOREE2 plasmid. The banana explants were incubated with A. tumefaciens cell suspension containing BLf- pOREE2 plasmid. Subsequently the corms with A. tumefaciens were incubated on solid medium in dark for three days.

The banana explants were transferred to suitable medium containing cefotaxime for regeneration.

The explants were sub cultured in medium containing the marker antibiotic, Kanamycin. The transformants were selected for screening i.e. DNA isolation from banana leaves followed by PCR using BLf primers.

The transformed banana plants after rooting and hardening were transferred to soil. Example 5

Production of Transgenic Milk:

With respect to the therapeutic relevance of the lactoferrin protein in biopharmaceutical industry, an experiment protocol has been designed for the production of transgenic milk with Vechur lactoferrin protein. Lf gene with a suitable promoter for overexpression is incorporated by electroporation or micro injection methods to the germ cells of a healthy breed of cow with good yield of milk, other than vechur. The objective of the study is to exploit the therapeutic and nutritional property of vechur lactoferrin protein by the development of a transgenic cow. Example 6:

The functional property of the Vechur cow B. Indicus Lf gene compared to other sps B. tarus by anti bacterial sensitivity assay for substantiating the interference of higher level arginine was done. It is evident from the study that Vechur Cow Lf protein has significant level of bactericidal property compared to the Lf protein of other breeds. This observation directly supports the fact that the arginine substitution noticed predominantly in Lf gene of Vechur cow has direct interference in enhancing its therapeutic function. Moreover it could be seen that the anti bacterial activity of Lf of Vechur cow is more than the synthetic antibiotic ampicillin (Figure 15).

Example 7:

The antibacterial sensitivity test (AST) conducted using different strains of E.coli, showed the inhibitory function of Lactoferrin of Vechur cow on bacterial growth is more significant than the Lf of other breeds.

As a control of the experiment, antibiotic penicillin & ampicillin were added. It is noticed that the action of Lf on suppressing bacterial growth was more efficient than the synthetic antibiotics. Quantitatively, it is recorded that suppression of bacterial growth in presence of antibiotic showed a value of 4cfu/ml where as the culture medium containing Lf exhibits an active suppression to the level of 1.9 cfu/ml. This reduction in bacterial growth noticed in the case of Lf compared to synthetic antibiotics clearly indicates, the unusual property of Lf i.e. absorption of iron from the bacterial growth medium besides the disintegration of the bacterial membranes similar to synthetic antibiotics. Thus, Lf has a synergistic effect in two ways ie absorption of iron from the medium and the disintegration of bacterial membrane on suppressing the bacterial growth unlike synthetic antibiotics. Since, iron is an essential factor for bacterial growth in the culture medium, its active absorption by Lf remains as a unique property of Lf.

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<210> 3

<21 1> 306

<212> DNA

<213> Bos indicus

<220>

<221> gene

<222> (1)..(306)

<400> 3

ggatccccag ccatgaagct cttcgtcccc gccctgctgt cccttggagc ccttggactg 60 tgtctggctg ccccgaggaa aaacgttcga tggtgtacca tctcccaacc tgagtggttc 120 aaatgccgcc gatggcagtg gaggatgaag aagctgggtg ctccctctat cacctgtgtg 180 aggagggcct ttgccttgga atgtatccgg gccatcgcgg agaaaaaggc ggatgctgtg 240 accctggatg gtggcatggt gtttgaggcg ggccgggacc cctacaaact gcggccagta 300 gcagca 306

<210> 4

<21 1> 48

<212> DNA

<213> Bos indicus

<220>

<221> gene

<222> (1)..(48)

<400> 4

ttcaaatgcc gccgatggca gtggaggatg aagaagctgg gtgctccc

<210> 5

<21 1> 16

<212> PRT

<213> Bos indicus

<220>

<221> PEPTIDE

<222> (1)..(16) <400> 5

Phe Lys Cys Arg Arg Tip Gin Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15

<210> 6

<21 1> 21

<212> D A

<213> Bos indicus

<220>

<221> prim transcript

<222> (1)..(21)

<223> Forward Primer 5'-3' <400> 6

ggatccccag ccatgaagct c 21

<210> 7

<21 1> 20

<212> DNA

<213> Bos indicus

<220>

<221> prim transcript

<222> (1)..(20)

<223> Reverse Primer 5'-3'

<400> 7

tgctgctact ggccgcagtt 20