KR20020061300A - Gene and protein of black goat rotavirus VP4 and VP7 - Google Patents

Abstract

PURPOSE: Provided are black goat rotavirus VP4 and VP7 genes and their proteins. The black goat rotavirus VP4 and VP7 genes are used for the diagnosis of and vaccine for rotavirus diarrhea. CONSTITUTION: VP4 and VP7 genes cloning comprises the steps of: extracting total RNA from black goat rotavirus; constructing cDNA; synthesizing a pair of primers specific to VP4 and VP7; performing PCR using cDNA as a template; sequencing PCR products and analyzing homology between the PCR products and each VP4 and VP7. The gene of black goat envelope protein VP4 has the nucleotide sequence of SEQ ID NO;1 and its protein has the amino acid sequence of SEQ ID No:2. The gene of black goat envelop protein VP7 has the nucleotide sequence of SEQ ID NO;3 and its protein has the amino acid sequence of SEQ ID NO;4.

Description

Genes and proteins of black goat rotavirus VP4 and VP7

The present invention relates to genes of VP4 and VP7 which are envelope proteins of the black goat rotavirus and proteins thereof.

Goats are ruminant mammals and are raised in large numbers in China, England, Europe, and North America. The primary purpose of goat breeding is primarily for milking, with the majority of goat milk being used to make cheese. In addition, young goats are used for food because they are very tender and have good taste, and species such as Angora and Cashmere are raised to obtain hair because they can make wool and cashmere. Young goats are also a source of kid's leather. In particular, goat breeding is more convenient than cattle in the tropics or in the greater part of the country for the large-scale milk production. In these countries, goat breeding is carried out on a large scale. It is a heavy industry, accounting for more than 20% of the country.

In the country where goat breeding is active, research on disease occurrence status and causes and finding preventive methods of livestock, including small and medium-sized ruminants, has been actively conducted since the early 70s, and most of them are respiratory and digestive diseases. For dual digestive diseases, according to a report by Munoz Escherichia coli (E. coli), salmonella (Salmonella), Clostridium (Clostridium), as well as bacteria and parasites such as Cryptosporidium (Cryptosporidium), rotavirus (rotavirus), coronavirus ( coronavirus ), etc. (Munoz, M. 1996, Role of enteric pathogens in the aetuology of neonatal diarrhea in lambs and goat kids in Spain. Epidemiol. Infect. 1172: 203-211). According to the report, in particular, rotavirus had a 6.1% incidence rate in Spain, but a 30-60% incidence rate in France and the United Kingdom.

Rotavirus is a genus of Reoviridae and causes diarrhea in many animals. Among the envelope proteins of rotavirus, VP7 is a glycosylated outermost protein and is a gene included in the rotavirus 6, 7, or 8th fragment. In addition, VP7 is the second most abundant protein after VP6 and a protective antigenic determinant capable of preventing rotavirus infection. VP4 is a dimer, which protrudes from the outer shell of Rotavirus with 60 short spikes. VP4 has biological functions such as neutralizing ability, hemagglutination, and cell adhesion in many viral lines, and is known to be involved in virulence determinant in mice and piglets.

Recently, about 670,000 domestic black goats are known to be raised in Korea. The number of heads of beef is 1/5 level of beef cattle, but as the domestic black goat is widely used for the purpose of rehabilitation, etc., it has increased more than three times in recent 5 years and the breeding size is also increasing. However, despite the increase in breeding, the previous studies on native black goats have been limited to the field for the establishment of good genetic seeds. It is not happening.

A recent distribution survey of black goat farms with more than 200 heads raised domestically showed 44% of respiratory diseases, 26% of digestive diseases, and 8% of papules and eye diseases. result). In particular, the seropositivity rate for rotavirus was found to be 79% or higher, indicating that rotavirus infection rate was considerably high. However, to date, studies on the black goat rotavirus are insignificant, and there are almost no research reports related to genetic analysis.

Accordingly, the inventors have conducted extensive research to analyze the nucleotide sequences of the VP4 and VP7 genes, which are envelope proteins of the virus, using the recently isolated black goat rotavirus, and the present invention has been completed based thereon.

It is therefore an object of the present invention to provide the base sequences of VP4 and VP7 which are envelope proteins of the virus.

Another object of the present invention is to provide amino acid sequences of VP4 and VP7 which are envelope proteins of the virus.

The base sequences of VP4 and VP7 for achieving the above object are represented by SEQ ID NO: 1, 3, respectively.

The amino acid sequences of VP4 and VP7 for achieving the above object are represented by SEQ ID NOs: 2 and 4, respectively.

Figures 1a, b, c, d, and e show the results of comparing the black goat rotavirus VP4 gene sequence with known C486, RRV and FRV-1,

Figure 2a and b shows the results of comparing the amino acid sequence of the black goat rotavirus VP4 with known C486, RRV and FRV-1,

3a and b show the results of comparing the black goat rotavirus VP7 gene sequence with known NCDV, SA11, and AU-1,

Figure 4 shows the results of comparative analysis of the black goat rotavirus VP7 amino acid sequence with known NCDV, SA11, and AU-1.

Hereinafter, the present invention will be described in more detail.

In the present invention, we cloned the genes of VP4 and VP7, which occupy most of the black goat rotavirus envelope protein isolated by the present inventors.

For cloning the VP4 gene, total RNA of black goat rotavirus was first extracted and then cDNA was prepared using the same. Next, a pair of primers specific for VP4 were synthesized using rotavirus sequences of known species, and PCR was then performed on the prepared cDNA as a template. As a result of PCR, a DNA fragment of 2,328 bp was obtained. As a result of sequencing and homology with VP4 sequencing of other species, homology of about 67 to 84% was shown. It was confirmed that (see Fig. 1a, b, c, d, e and 2a, b). Sequencing revealed that the VP4 gene had a base of 2,328 bp, identical to bovine rotavirus C486 (Genbank accession No. Y00127) and monkey rotavirus RRV (Genbank accession No. M18736), and cat rotavirus FRV-1 (Genbank). accession No. D10971). The sequence of the VP4 gene is represented by SEQ ID NO: 1. The amino acid sequence of VP4 prepared based on the nucleotide sequence was shown in SEQ ID NO: 2 and has a length of 776.

Cloning of the VP7 gene was performed in the same manner as in the case of VP4, and a 978 bp DNA fragment was obtained as a PCR result. As a result of sequence analysis and homology with the VP7 sequences of other species, homology of about 67 to 85% was shown, confirming that the DNA fragment was the black goat Rotavirus VP7 gene. Sequencing showed that the VP7 gene had a base of 978 bp, which was bovine rotavirus NCDV (Genbank accession No. M63266), monkey rotavirus SA11 (Genbank accession No. AH003158), and human rotavirus AU-1 (Genbank accession No. D89873). The sequence of the VP7 gene is represented by SEQ ID NO: 3. The amino acid sequence of VP7 prepared based on the nucleotide sequence was shown in SEQ ID NO: 4 and has a length of 326.

The p4 and VP7 genes obtained above were compared with the sequences of other known Rotavirus strains for determination of p type.

As a result, VP4 showed the highest homology of 83/91% with RRV derived from type 3, and VP7 also had a rodent-derived Eb, human-derived HCR3, monkey-derived RRV, and monkey-derived SA11 with 76-85. The black goat rotavirus isolated with the highest homology (/ 81-95%) was found to be the most similar to the type 3 close to the human-derived type.

In the present invention, the cloned VP4 and VP7 genes can be put into an expression vector and then overexpressed to obtain a protein, which can be used to produce antibodies to rotaviruses, and can be used in the production of diagnostic kits such as DNA chips or protein chips. In addition, it can be used as a DNA vaccine, and can be used for other purposes, which contributes to the prevention and treatment of diarrheal diseases caused by rotavirus.

In addition, the characteristics of the VP4 and VP7 genes cloned in the present invention is shown to be closer to the type derived from human than ruminants, and can be used as useful data for humans as well as black goats.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1

Isolation of Black Goat Rotavirus

Severe diarrhea from farms around the country, where more than 200 black goats are bred, or dead black goats were selected due to diarrhea, and small intestine sections, feces and serum were collected and stored at -70 ℃.

Cell line TF104, a monkey kidney cancer cell, was obtained from the National Veterinary Research and Quarantine Service, and then α-MEM (Gibco BRL Co. Cultured on) was prepared.

The collected black goat specimen was suspended 1: 5 in PBS, filtered through a 0.2 μm filter, sensitized in 10 μg / ml (final concentration) trypsin, and then incubated in a 5% CO ₂ , 37 ° C. incubator for 1 hour. One cell was adsorbed to infect the host cell TF104. Infected cells were washed three times with PBS and incubated in a-MEM with 0.5 μg / ml trypsin until the cytopathic effect. If degeneration occurs after 48 to 72 hours after infection, the cells are collected, frozen and thawed three times, and then centrifuged at 4000 × g for 15 minutes to collect the supernatant containing virus and stored at -70 ℃. It was.

Indirect fluorescent antibody method using monoclonal antibody was used to confirm the presence of rotavirus in the small intestine tissues and cells. In order to identify rotavirus present in the specimen tissue, frozen intestinal tissue stored at −20 ° C. was first sliced using a microtome, superimposed on slide glass, and dried. The tissues were then fixed with acetone for 5 minutes and first reacted with monoclonal antibodies against bovine rotavirus VP8 at room temperature for 1 hour. After completion of the first reaction, the tissues were washed three times with PBS three times, and then reacted with anti-mouse IgG attached to the FITC for 1 hour. Microscopic observation confirmed the virus infection.

To confirm the presence of intracellular rotavirus, first put a sterile cover slip at the bottom of the 6-well plate and float TF104 cells in a culture medium containing 10% FBS (Fetal Bovine Serum) to form a cell monolayer. It was. After the monolayer cells were formed, the cells were washed three times with physiological saline, and then infected with 500 µl of rotavirus solution isolated from the black goat specimen for 1 hour, followed by incubation until cytopathic effect appeared. When the cytopathic effect appeared, the cells were washed three times with PBS and dried overnight. The dried cells were fixed with acetone for 5 minutes and then reacted with monoclonal antibodies against bovine rotavirus VP8 and anti-mouse IgG attached with FITC in the same manner as described above and observed by fluorescence microscopy (FIGS. 1A, b, c, d, e).

As a result, it was confirmed that the TF104 cells were infected with rotavirus, and as a result, the virus isolated from the black goat specimen was rotavirus.

Example 2

Black Goat Rotavirus VP4 Gene Sequencing

Trizol (GIBCO) was used to isolate the total RNA from the black goat rotavirus obtained in Example 1 using the protocol of the manufacturer's protocol, using reverse transcriptase as a known method using the isolated RNA as a template. CDNA was synthesized (Sambrook et al., Molecular cloning, 1989). Using 250 ng of the cDNA as a template, 20 pmol each of the forward primer represented by SEQ ID NO: 5 and the reverse primer represented by SEQ ID NO: 6 specific for Rotavirus VP4 were used as PCR buffers (dNTP 2.5 mM, 2.5 units of polymerase 2.5, 10). PCR was performed on polymerase buffer (500 mM KCl, 100 mM TrisCl, 15 mM MgCl _2, 0.1% gelatin). The PCR was performed using a PCR cycler (Perkin Elmer) for 5 minutes at 94 ° C. first, followed by 20 times at 94 ° C., 30 seconds at 60 ° C., 30 seconds at 72 ° C., and finally 30 times. 10 minutes was carried out at 72 ℃ to obtain the result. The resultant was electrophoresed on 0.9% agarose gel to confirm that a DNA fragment having a size of 2,328 bp was obtained. The amplified fragment was cloned into a pGEM-T (Promega) vector to obtain a pGEM-VP4 vector.

In order to confirm whether the cloned DNA fragment was a VP4 gene, the pGEM-VP4 vector was commissioned to a basic science research institute and sequenced to obtain a nucleotide sequence represented by SEQ ID NO: 1. The obtained sequences were compared and analyzed with other known VP4 gene sequences, and as a result, 2,328 bp at base level, identical to bovine rotavirus C486 (Genbank accession No. Y00127) and monkey rotavirus RRV (Genbank accession No. M18736). It was found that there are three more bases than the cat rotavirus FRV-1 (Genbank accession No. D10971). The amino acid sequence prepared based on the nucleotide sequence was represented by SEQ ID NO: 2 and had a length of 776.

Comparative analysis of the sequences of VP4 obtained in this example with known C486, RRV and FRV-1 showed 75-84% homology and 79-91% homology with the nucleotide sequence. 1a, b, c, d, e and 2a, b).

Example 3

Black Goat Rotavirus VP7 Gene Sequencing

RNA was extracted in the same manner as in Example 2, cDNA was synthesized, and then the same conditions as in Example 2 were performed using the forward primer represented by SEQ ID NO: 7 and the reverse primer represented by SEQ ID NO: 8 specific for Rotavirus VP7. PCR was performed at. PCR was performed using a PCR cycler (Perkin Elmer) for 5 minutes at 94 ° C. first, followed by 20 times at 94 ° C., 30 seconds at 60 ° C., 30 seconds at 72 ° C., and finally 72 times. 10 minutes was carried out to obtain the result. The resultant was subjected to electrophoresis on 0.9% agarose gel to confirm that a 978 bp DNA fragment was obtained. The amplified fragment was cloned into a pGEM-T (Promega) vector to obtain a pGEM-VP7 vector. In order to confirm that the cloned DNA fragment was VP7 gene, the pGEM-VP4 vector was commissioned to a basic science research institute and sequenced. As a result, a nucleotide sequence represented by SEQ ID NO: 3 was obtained. The obtained sequences were compared with other known VP7 gene sequences, resulting in bovine rotavirus NCDV (Genbank accession No. M63266), monkey rotavirus SA11 (Genbank accession No. AH003158), and human at 978 bp in basal level. It was identical to rotavirus AU-1 (Genbank accession No. D89873) and was found to have three more bases than feline virus FRV-1 (Genbank accession No. D10971). In addition, the amino acid sequence was prepared based on the nucleotide sequence, and the amino acid sequence represented by SEQ ID NO: 4 was obtained. It was confirmed that NCDV, SA11, and AU-1 all had the same length of 326.

Comparative analysis of the VP7 sequence obtained in this example with the known NCDV and SA11 showed 77-83% homology and 85-95% homology with the nucleotide sequence (FIGS. 3A and B). And 4).

Example 4

P-type Determination of Black Goat Rotavirus VP4

In order to determine the degree of homology between the nucleotide sequence and amino acid sequence of the black goat Rotavirus VP4, the sequences represented by SEQ ID NOs: 1 and 2 were respectively derived from cows derived from NCDV, UK, B223, and 993/83, pigs. Sequence homology with VP4 of Rotavirus isolates such as OSU, and MDR13, monkey-derived RRV, human-derived AU-1, SA11, 1076, K8, 69M, Mc35, and HAL1166, horse-derived L338, rodent-derived Eb, sheep-derived Lp14 Compared.

As a result, as shown in Table 1, 71/75% (base homology / amino acid homology) with NCDV of type 1, 79/88% with SA11 belonging to type 2, and 83/91% with RRV belonging to type 3 Homologous, 72/71% with L26 in type 4, 72/76% with UK in type 5, 72/72% with 1076 in type 6 and 75/80 with OSU in type 7. %, 72/71% with Wa belonging to type 9, 70/69% with K8, 76/85% with 69M belonging to type 10, 60/81% with B223 belonging to type 11, B2 belonging to type 12 75/81% with MDR13 belonging to type 13, 75/78% with MDR13 belonging to type 13, 70/70% with Mc35 belonging to type 14, 76/82% with Lp14 belonging to type 15, 71/76% with Eb belonging to type 16 , 67/63% with 993/83 belonging to type 17, 70/71% with HAL1166 belonging to type 18, and 75/78% homology with L338 belonging to type 19.

Finally, the homology was found to be in the range of 83-60% for the nucleotide sequence and 91-69% for the amino acid sequence. In particular, it showed the highest homology of 83/91% with the RRV belonging to type 3 Rotavirus was found to be the most similar to type 3.

Strain VP4 genotype P antigen type % Sequence Homology Nucleic acid amino acid NCDV One P6 71 75 SA11 2 P6 79 88 RRV 3 P5 83 91 L26 4 72 71 UK 5 P7 72 76 1076 6 P2A 72 72 OSU 7 P9 75 80 Wa 8 P1A 72 71 K8 9 P3A 70 69 69M 10 P4 76 85 B223 11 P8 60 60 H2 12 75 81 MDR13 13 75 78 Mc35 14 P3B 70 70 Lp14 15 76 82 Eb 16 P10 71 76 993/83 17 67 63 HAL1166 18 P11 70 71 L338 19 75 78

Example 5

P-type Determination of Black Goat Rotavirus VP7

In order to determine the degree of homology between the nucleotide sequence and amino acid sequence of the black goat Rotavirus VP4, the sequences represented by SEQ ID NOs: 3 and 4 were respectively derived from bovine-derived NCDV, KK3, pig-derived OSU, YM, and monkey-derived RRV. Sequence homology with rotavirus isolates such as SA11, human-derived Wa, Gottfried, HCR3, horse-derived L338, F123, rodent-derived Eb, and chicken-derived Ch2 was compared.

As a result, as shown in Table 1, 75/81% with Wa belonging to type 1, 75/75% with S2 belonging to type 2, 77/89% with Eb belonging to type 3, 85/95% with HCR3 and RRV. 76/81%, 83/95% with SA11, 75/78% with Gottfried belonging to type 4, 78/85% with OSU belonging to type 5, 77/85% with NCDV belonging to type 6, type 7 67/59% with Ch2, 75/80% with B37 belonging to type 8, 77/85% with 116E belonging to type 9, 77/83% with KK3 belonging to type 10, 78 / with YM belonging to type 11 88%, 75/81% with L26 belonging to type 12, 77/82% with L338 belonging to type 13, and 83/87% homology with F123 belonging to type 14.

Finally, the homology was found to be in the range of 83-60% for the nucleotide sequence and 91-69% for the amino acid sequence, especially 76-85 / 81-95 with Eb, HCR3, RRV, and SA11 belonging to type 3 Black goat rotavirus isolated with the highest homology in% was found to be the most similar to type 3.

Strain genotype % Sequence Homology Nucleic acid amino acid Wa One 75 81 S2 2 75 75 Eb 3 77 89 HCR3 3 85 95 RRV 3 76 81 SA11 3 83 95 Gottfried 4 75 78 OSU 5 78 85 NCDV 6 77 85 Ch2 7 67 59 B37 8 75 80 116E 9 77 85 KK3 10 77 83 YM 11 78 88 L26 12 75 81

Since the base sequence and amino acid sequence of the rotavirus envelope protein VP4 and VP7 gene according to the present invention can be used for the diagnosis and vaccine development of black goat rotavirus diarrhea, it is expected to improve the productivity of domestic black goat breeding industry.

Claims (4)

Black goat rotavirus envelope protein VP4 gene having a nucleotide sequence represented by SEQ ID NO: 1. Black goat rotavirus envelope protein VP4 having the amino acid sequence represented by SEQ ID NO: 2. Black goat rotavirus envelope protein VP7 gene having a nucleotide sequence represented by SEQ ID NO: 3. Black goat rotavirus envelope protein VP7 having the nucleotide sequence represented by SEQ ID NO: 4.