CN111635910A - Panda rotavirus CH-1 strain VP7 positive plasmid and construction method and application thereof - Google Patents
Panda rotavirus CH-1 strain VP7 positive plasmid and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a giant panda rotavirus CH-1 strain VP7 positive plasmid and a construction method and application thereof; relates to the technical field of giant panda rotavirus detection, and the VP7 positive plasmid is constructed by the VP7 gene and a PUC57 vector; the VP7 positive plasmid is transformed into competent cells, and the plasmid is cultured and extracted by the cells, so that the VP7 positive plasmid can be infinitely obtained, and PCR detection of panda rotavirus can be realized in basic units and units incapable of culturing panda rotavirus CH-1 strain viruses.
Description
Technical Field
The invention relates to the technical field of panda rotavirus detection, in particular to a panda rotavirus CH-1 strain VP7 positive plasmid and a construction method and application thereof.
Background
Rotavirus is one of the major pathogens causing non-bacterial diarrhea in infants and various young animals; the Wangdong is equal to 2009, and Rotavirus is firstly detected and separated from diarrhea excrement of pandas and is named as a Giant Panda Rotavirus (GPRV) CH-1 strain. The GPRV CH-1 strain is mainly used for infecting weaned giant pandas of 5-11 months old, and generally shows symptoms such as body temperature rise, vomiting, watery diarrhea, frankliniella-like diarrhea, total blood and leucocyte in diarrhea and neutrophilia, lymphocyte reduction, ascites, refractory flatulence, diarrhea lingering and even death and the like after infection, the treatment course of the disease can be greatly shortened through conventional symptomatic treatment, the cure rate reaches 100%, complications rarely occur, but no effective vaccine or special-effect medicine is used for preventing and treating the GPRV infection at present, and serious threat is caused to the health of the giant pandas.
The content of the GPRV CH-1 strain in the panda feces is high, and molecular biological diagnosis, namely conventional RT-PCR and qRT-PCR, can be carried out by extracting the total RNA of the panda feces. Designing a primer according to the sequence of a conserved gene VP7 of a GPRV CH-1 strain, carrying out reverse transcription on RNA extracted from panda feces and RNA of virus liquid preserved in a laboratory simultaneously, carrying out PCR amplification on the obtained cDNA, carrying out agarose gel electrophoresis detection on a product amplified by RT-PCR and sequencing, wherein the product can be used for detecting about 1pg of virus RNA at least, and the diagnostic result has good repeatability and can be used for clinical rapid diagnosis of the GPRV CH-1 strain.
In addition, primers are designed according to the gene sequence of the VP4 of the GPRV CH-1 strain for qRT-PCR amplification, the lowest detectable copy number is 1.0 multiplied by 100 copies/mu l, compared with the conventional RT-PCR, the sensitivity is at least 100 times higher, and the specificity and the repeatability of the two methods are better.
However, positive controls are involved in molecular biological diagnosis, and the positive control of the current PCR detection is obtained by culturing a GPRVCH-1 virus strain, extracting RNA, performing reverse transcription, and obtaining cDNA as the positive control; and for wild animal breeding units such as a lot of pandas and the like, no equipment and technical conditions are provided for culturing the GPRV CH-1 virus strain, so that a positive control required in PCR detection cannot be obtained, and the popularization and application of RT-PCR in basic units are limited.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a giant panda rotavirus CH-1 strain VP7 positive plasmid, which can infinitely obtain VP7 positive plasmid by transforming the VP7 positive plasmid into host cells, and then carrying out cell culture and plasmid extraction, and can realize the PCR detection of the giant panda rotavirus in basic units and units which can not carry out the culture of the giant panda rotavirus CH-1 strain virus.
In view of the above, the first aspect of the present invention provides a panda rotavirus CH-1 strain VP7 positive plasmid, wherein the VP7 positive plasmid is constructed by the VP7 gene and a PUC57 vector.
The GPRV CH-1 strain belongs to rotavirus of reoviridae, and virus particles are spherical and similar to a wheel shape and have the diameter of 70 nm. The genome of the GPRV CH-1 strain is linear double-strand RNA, which comprises 11 gene segments, wherein the genes respectively encode 6 structural proteins (VP1-VP4, VP6 and VP7) and 5 non-structural proteins (NSP1-NSP5), and the lengths are 3287bp, 2717bp, 2591bp, 2362bp, 1356bp, 1042bp, 1532bp, 1042bp, 1017bp, 750bp and 663bp in sequence; wherein VP4, VP6 and VP7 are main antigenic genes of GPRV.
Research shows that the structural protein constructs the whole virus particle and is divided into VP1, VP2, VP3, VP4, VP6 and VP 7; nonstructural proteins (NSP) are produced only in rotavirus infected cells and do not constitute virions; VP7 is main glucoprotein and neutralization antigen of rotavirus shell, determines its serotype, VP7 is from 7 th, 8 th or 9 th gene (depending on different strains), the total length of coding gene is 1062bp, the molecular weight is 37ku, it accounts for 30% of total amount of virus protein, it can induce independently to produce neutralizing antibody, plays an important role in inducing immunity; therefore, the VP7 gene is selected as a key gene for detecting rotavirus, and has good representativeness and stability.
The VP7 gene sequence of the GPRV CH-1 strain is a giant panda rotavirus CH-1 strain VP7 gene sequence which is inquired and obtained in the National Center for Biotechnology Information (NCBI) of the United states and has the accession number GU188284, the length is 1042bp, and the gene sequence is shown as SE Q ID NO: 1 is shown in the specification; the method specifically comprises the following steps:
1 CCGTCTGGCT AGCGGTTAGC TCCTTTTAAT GTATGGTATT GAATATACCA CAATTCTAAT
61 CTTTCTGATA TCAATCATTC TATTCAATTA TATATTAAAG TCAGTAACTC GAACGATGGA
121 CTATATTATT TACAGATTTC TATTAATAAC GGCAGCACTA CTTGCATTTA CAAAAGCACA
181 GAATTACGGA ATTAATCTAC CTATAACAGG ATCAATGGAT ACTGCATACG CTAATTCAAC
241 TCAAGAAGAA ACATTCTTGA CATCTACATT ATGCTTATAT TATCCGACTG AAGCGAGTAA
301 TCAAATAAAC GATGGTGAAT GGAAAGACAC GTTATCTCAA ATGTTTCTTA CAAAAGGATG
361 GCCGACAGGA TCAGTTTATT TTAAAGAATA CTCAAGTATC GTAGACTTCT CAGTCGATCC
421 ACAATTATAC TGTGATTACA ATTTGGTACT AATGAAATAT GATCAAAATC TTGAATTAGA
481 TATGTCAGAG TTAGCTGATT TAATACTGAA TGAATGGCTA TGTAATCCAA TGGACATAAC
541 TTTATATTAT TATCAACAGA CAGGAGAATC AAATAAATGG ATATCAATGG GATCATCATG
601 TACTATTAAA GTTTGTCCAC TGAATACACA GACACTAGGA ATAGGCTGTC AAACGACGCA
661 TGTAGACTCA TTTGAGATCG TTGCTGAAAA TGAAAAACTA GCTATAGTGG ATGTCGTTGA
721 TGGCATAGAT CATAAAATAA ATTTAACAAC TACTACATGC ACAATTCGAA ATTGTAAGAA
781 GCTGGGACCT AGAGAAAACG TAGCTGTAAT ACAGGTTGGA GGCTCTGATA TATTAGATAT
841 TACAGCAGAT CCAACGACCA ATCCACAGAC TGAGAGAATG ATGAGAGTGA ATTGGAAAAA
901 GTGGTGGCAA GTTTTTTATA CAATAGTTGA TTATATTAAT CAAATTGTGC AAGTAATGTC
961 CAAAAGATCA CGATCGCTAA ACGCAGCAGC CTTCTATTAT AGAGTATAGA TATATCTTAG
1021 ATTAGAATTG TATGATGTGA CC；
the VP7 positive plasmid is transformed into a host cell, and the VP7 positive plasmid can be infinitely obtained through cell culture and plasmid extraction, so that PCR detection of panda rotavirus can be realized in basic units and units incapable of culturing panda rotavirus CH-1 strain viruses.
Preferably: the two ends of the VP7 gene sequence are designed with BamHI/SalI enzyme cutting sites; specifically, the VP7 gene sequence after BamHI/SalI cleavage site is designed as SE Q ID NO: 2 is shown in the specification; the method specifically comprises the following steps:
1 GTCGAC CCGTCTGGCT AGCGGTTAGC TCCTTTTAAT GTATGGTATT GAATATACCA
57 CAATTCTAAT CTTTCTGATA TCAATCATTC TATTCAATTA TATATTAAAG TCAGTAACTC
117 GAACGATGGA CTATATTATT TACAGATTTC TATTAATAAC GGCAGCACTA CTTGCATTTA
177 CAAAAGCACA GAATTACGGA ATTAATCTAC CTATAACAGG ATCAATGGAT ACTGCATACG
237 CTAATTCAAC TCAAGAAGAA ACATTCTTGA CATCTACATT ATGCTTATAT TATCCGACTG
297 AAGCGAGTAA TCAAATAAAC GATGGTGAAT GGAAAGACAC GTTATCTCAA ATGTTTCTTA
357 CAAAAGGATG GCCGACAGGA TCAGTTTATT TTAAAGAATA CTCAAGTATC GTAGACTTCT
417 CAGTCGATCC ACAATTATAC TGTGATTACA ATTTGGTACT AATGAAATAT GATCAAAATC
477 TTGAATTAGA TATGTCAGAG TTAGCTGATT TAATACTGAA TGAATGGCTA TGTAATCCAA
537 TGGACATAAC TTTATATTAT TATCAACAGA CAGGAGAATC AAATAAATGG ATATCAATGG
597 GATCATCATG TACTATTAAA GTTTGTCCAC TGAATACACA GACACTAGGA ATAGGCTGTC
657 AAACGACGCA TGTAGACTCA TTTGAGATCG TTGCTGAAAA TGAAAAACTA GCTATAGTGG
717 ATGTCGTTGA TGGCATAGAT CATAAAATAA ATTTAACAAC TACTACATGC ACAATTCGAA
777 ATTGTAAGAA GCTGGGACCT AGAGAAAACG TAGCTGTAAT ACAGGTTGGA GGCTCTGATA
837 TATTAGATAT TACAGCAGAT CCAACGACCA ATCCACAGAC TGAGAGAATG ATGAGAGTGA
897 ATTGGAAAAA GTGGTGGCAA GTTTTTTATA CAATAGTTGA TTATATTAAT CAAATTGTGC
957 AAGTAATGTC CAAAAGATCA CGATCGCTAA ACGCAGCAGC CTTCTATTAT AGAGTATAGA
1017 TATATCTTAG ATTAGAATTG TATGATGTGA CCGGATCC。
preferably, the VP7 gene after designing BamHI/SalI cleavage site can be obtained by artificial synthesis.
The second aspect of the present invention provides a method for constructing the VP7 positive plasmid, including the following steps:
respectively treating the VP7 gene and the PUC57 vector by using BamHI/SalI enzyme;
carrying out nucleic acid electrophoresis analysis on the processed product and recovering 1048bp and 2697bp gene sequences;
and (3) connecting the 1048bp gene sequences with the 2697bp gene sequences to obtain the VP7 positive plasmid.
Preferably, the treatment of the VP7 gene and the PUC57 vector with BamHI/SalI enzyme, respectively, comprises:
incubating reaction systems respectively containing the VP7 gene and the PUC57 vector at 37 ℃ for 3.5-5 h;
wherein the reaction system comprises 2ul BamHI/SalI enzyme (2 ul BamHI/SalI enzyme can cut 2ug of gene according to the instruction, 2ul BamHI/SalI enzyme can ensure enough enzyme), 10ul 10 × buffer, 10ul VP7 gene/PUC 57 vector and 33ul ddH2And O, the total amount is 50ul (the system is a common system) (the specific use amount of each component is determined according to the VP7 gene/PUC 57 vector and the cutting efficiency of enzyme, and generally, the amount of the cut gene or plasmid is not less than 1 ug). Tong (Chinese character of 'tong')The cutting rate of BamHI/SalI enzyme can be effectively improved by the restriction of each component in the system.
Preferably, the connecting 1048bp and 2697bp gene sequences comprises:
incubating the reaction system containing 1048bp and 2697bp gene sequences overnight at 4 ℃, and then incubating for 3.5-4.5 h at 15-17 ℃;
wherein, the reaction system comprises: 1ul T4DNA Ligase (the specific dosage used is determined according to the instructions), 1ul 10 XBuffer, 6ul VP7 gene and 2ul PUC57 vector, totaling 10ul system (the ratio of inserted gene fragment and vector should be about 3: 1).
The third aspect of the present invention provides an Escherichia coli strain containing the VP7 positive plasmid or the VP7 positive plasmid constructed by the above construction method.
Preferably, the Escherichia coli strain is obtained by transforming the VP7 positive plasmid or the VP7 positive plasmid constructed by the construction method into Escherichia coli TOP10 competent cells.
The fourth aspect of the invention provides an application of the VP7 positive plasmid or the VP7 positive plasmid constructed by the construction method as a positive control in PCR detection of panda rotavirus.
The fifth aspect of the invention provides a PCR detection kit for detecting panda rotavirus, which comprises the VP7 positive plasmid or the VP7 positive plasmid constructed by the construction method.
The invention also provides a PCR detection kit for detecting panda rotavirus, which comprises a standard substance, wherein the standard substance is VP7 positive plasmid extracted from the escherichia coli strain.
The invention has the beneficial effects that:
the VP7 positive plasmid is transformed into competent cells, and the plasmid is cultured and extracted by the cells, so that the VP7 positive plasmid can be infinitely obtained, and PCR detection of panda rotavirus can be realized in basic units and units incapable of culturing panda rotavirus CH-1 strain viruses.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 is a drawing of agarose gel electrophoresis provided in example 3 of the present invention;
FIG. 2 shows the result of gene sequence alignment provided in example 3 of the present invention;
FIG. 3 is an agarose gel electrophoresis image provided in example 4 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
The raw materials and equipment used in the following examples were as follows:
BamHI/SalI endonuclease: purchased from Thermo Fisher Scientific;
VP7 gene designed with BamHI/SalI cleavage site: gene sequences such as SE Q ID NO: as shown in figure 2, the first and second,
synthesized by Biotechnology corporation;
PUC57 vector: no. 181215PA5661-10R16, 2710bp in size, and carrying ampicillin resistance gene, purchased from Thermo Fisher Scientific;
t4DNA Ligase: purchased from Thermo Fisher Scientific;
coli TOP10 competent cells: purchased from Thermo Fisher Scientific;
a Tiangen gum recovery kit: commodity number DP 209-03; tiangen Biochemical technology (Beijing) Ltd.
Example 1
Giant panda rotavirus CH-1 strain VP7 positive plasmid
The VP7 positive plasmid is constructed by the VP7 gene and the PUC57 vector;
wherein, the two ends of the gene sequence of VP7 are designed with BamHI/SalI enzyme cutting sites.
Second, construction method
The construction method of the VP7 positive plasmid comprises the following steps:
the reaction systems containing the VP7 gene and the PUC57 vector, respectively, were incubated at 37 ℃ for 4 hours, wherein the reaction systems included 2ul BamHI/SalI enzyme, 10ul 10 × buffer, 10ul VP7 gene/PUC 57 vector, and 33ul ddH2O；
Performing nucleic acid electrophoresis on the incubated product on 1% agarose gel at 150V for 30min, and recovering 1048bp and 2697bp gene sequences on the adhesive tape by using a tiangen gel recovery kit;
and (2) incubating a reaction system containing 1048bp and 2697bp gene sequences at the temperature of 4 ℃ for overnight, wherein the reaction system comprises: 1ul of T4DNA Ligase, 1ul of 10 Xbuffer, 6ul of VP7 gene and 2ul of PUC57 vector, and then incubating for 3.5-4.5 h at 15-17 ℃ to obtain the VP7 positive plasmid.
Example 2
One, Escherichia coli strain containing VP7 positive plasmid in example 1;
the above E.coli strain was obtained by transforming the VP7 positive plasmid of example 1 into E.coli TOP10 competent cells.
Second, transformation method
The method for transforming the VP7 positive plasmid into escherichia coli TOP10 competent cells comprises the following steps:
1ul of the VP7 positive plasmid solution (the content is not more than 50ng) constructed in the example 1 is added into 50ul of escherichia coli TOP10 competent cell suspension, and ice bath is carried out for 30min after the mixture is gently mixed;
then, the mixture is subjected to heat shock for 90s in a water bath at 42 ℃, and is quickly placed on ice for 2min, and the bacteria liquid is not required to be oscillated in the whole process; adding 700 mu lLB liquid culture medium (without antibiotic), mixing uniformly, shaking and culturing in a shaker at 37 ℃ and 180rpm for 40min to restore the bacteria to normal growth state and express antibiotic resistance gene coded by plasmid;
and (3) shaking the bacterial liquid uniformly, coating the bacterial liquid on an LB agar plate containing 100ug/ml ampicillin, placing the bacterial liquid for half an hour with the front side facing upwards, after the bacterial liquid is completely absorbed by the culture medium, carrying out inverted culture at 37 ℃ for 14 hours, picking a single bacterial colony to an LB liquid culture medium containing 100ug/ml ampicillin resistance, and culturing the single bacterial colony at 180rpm and 37 ℃ until the culture medium is turbid to obtain the Escherichia coli strain containing the VP7 positive plasmid.
Example 3
Collecting Escherichia coli strains containing VP7 positive plasmids obtained by culturing in example 2, extracting bacterial plasmids by using a Tiangen plasmid extraction kit (catalog number DP118), measuring the concentration of the extracted plasmids by using an Onedrop spectrophotometer, wherein the concentration of the extracted plasmids is 160 ng/ul, shearing the plasmids by using a-BamHI/SalI restriction endonuclease, performing gel electrophoresis analysis and sequencing, recovering gel strips at 1048bp of gel electrophoresis, extracting DNA, sequencing, and comparing with a VP7 gene sequence (accession number GU188284) of a panda rotavirus CH-1 strain; the results of gel electrophoresis are shown in FIG. 1, and the results of partial alignment are shown in FIG. 2;
as can be seen from FIGS. 1 and 2, the homology of the gene sequence at 1048bp of gel electrophoresis and the gene sequence of VP7 of panda rotavirus CH-1 strain is 100%, thus the extracted plasmid contains the gene of VP7 of panda rotavirus CH-1 strain; two bands appear at 1048bp and 2697bp of the plasmid after enzyme digestion, which indicates that the extracted plasmid is a VP7 positive plasmid containing a VP7 gene and a PUC57 vector.
Example 4
Verification of VP7 positive plasmid as positive control:
obtaining VP7 positive plasmid extracted from Escherichia coli strain containing VP7 positive plasmid in example 3;
comparing the gene sequence of VP7 of panda rotavirus CH-1 strain with the gene sequence of canine rotavirus CU-1 strain VP7, and designing a panda rotavirus primer according to the comparison result, wherein the primer sequence is shown as SE Q ID NO: 3 and SE Q IDNO: 4, wherein, SE Q ID NO: 3 is 5'-TGGTATTGAATATACCACAAT-3'; SE Q ID NO: 4 is 5'-AAGGATGGCCGACAGGATCAG-3', the size of the amplified product is 340 bp;
taking physiological saline as a negative control, taking panda rotavirus PCR positive control (which is a gift from professor of beauty), and taking VP7 positive plasmid as a sample to be verified for PCR amplification; the PCR reaction adopted a 20ul system: 10uL of 2 xTaq PCRmastermix, 6uL of sterilized double distilled water, 3uL of reverse transcription product, and 0.5 uL of each of upstream primer and downstream primer; amplification was performed under the following conditions: pre-denaturation at 95 deg.C for 5min, cyclic denaturation at 95 deg.C for 30s, annealing renaturation at 55 deg.C for 30s, extension at 72 deg.C for 30s, 30 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C; taking the amplification product, carrying out 10g/L agarose gel electrophoresis for 30min under the condition of 110V, and observing the result; the results of gel electrophoresis are shown in FIG. 3, in which 1 is physiological saline, 2 is VP7 positive plasmid, and 3 is positive control;
as can be seen from FIG. 3, 1 did not appear any band as a negative control; 3 single and bright strip appears at 340bp, which shows that the designed rotavirus primer can be used for detecting panda rotavirus; 2 shows a single bright band at 340bp, which indicates that the VP7 positive plasmid can be used as a positive control in PCR detection of panda rotavirus like 3.
The above is only a partial experimental result in a large number of studies by the applicant, but it can be stated that:
the VP7 positive plasmid is transformed into competent cells, and the plasmid is cultured and extracted by the cells, so that the VP7 positive plasmid can be infinitely obtained, and PCR detection of panda rotavirus can be realized in basic units and units incapable of culturing panda rotavirus CH-1 strain viruses.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
SEQUENCE LISTING
<110> research base for breeding pandas in Chengdu province
<120> giant panda rotavirus CH-1 strain VP7 positive plasmid and construction method and application thereof
<130>2020
<160>4
<170>PatentIn version 3.3
<210>1
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ccgtctggct agcggttagc tccttttaat gtatggtatt gaatatacca caattctaat 60
ctttctgata tcaatcattc tattcaatta tatattaaag tcagtaactc gaacgatgga 120
ctatattatt tacagatttc tattaataac ggcagcacta cttgcattta caaaagcaca 180
gaattacgga attaatctac ctataacagg atcaatggat actgcatacg ctaattcaac 240
tcaagaagaa acattcttga catctacatt atgcttatat tatccgactg aagcgagtaa 300
tcaaataaac gatggtgaat ggaaagacac gttatctcaa atgtttctta caaaaggatg 360
gccgacagga tcagtttatt ttaaagaata ctcaagtatc gtagacttct cagtcgatcc 420
acaattatac tgtgattaca atttggtact aatgaaatat gatcaaaatc ttgaattaga 480
tatgtcagag ttagctgatt taatactgaa tgaatggcta tgtaatccaa tggacataac 540
tttatattat tatcaacaga caggagaatc aaataaatgg atatcaatgg gatcatcatg 600
tactattaaa gtttgtccac tgaatacaca gacactagga ataggctgtc aaacgacgca 660
tgtagactca tttgagatcg ttgctgaaaa tgaaaaacta gctatagtgg atgtcgttga 720
tggcatagat cataaaataa atttaacaac tactacatgc acaattcgaa attgtaagaa 780
gctgggacct agagaaaacg tagctgtaat acaggttgga ggctctgatatattagatat 840
tacagcagat ccaacgacca atccacagac tgagagaatg atgagagtga attggaaaaa 900
gtggtggcaa gttttttata caatagttga ttatattaat caaattgtgc aagtaatgtc 960
caaaagatca cgatcgctaa acgcagcagc cttctattat agagtataga tatatcttag 1020
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gtcgacccgt ctggctagcg gttagctcct tttaatgtat ggtattgaat ataccacaat 60
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gatggactat attatttaca gatttctatt aataacggca gcactacttg catttacaaa 180
agcacagaat tacggaatta atctacctat aacaggatca atggatactg catacgctaa 240
ttcaactcaa gaagaaacat tcttgacatc tacattatgc ttatattatc cgactgaagc 300
gagtaatcaa ataaacgatg gtgaatggaa agacacgtta tctcaaatgt ttcttacaaa 360
aggatggccg acaggatcag tttattttaa agaatactca agtatcgtag acttctcagt 420
cgatccacaa ttatactgtg attacaattt ggtactaatg aaatatgatc aaaatcttga 480
attagatatg tcagagttag ctgatttaat actgaatgaa tggctatgta atccaatgga 540
cataacttta tattattatc aacagacagg agaatcaaat aaatggatat caatgggatc 600
atcatgtact attaaagttt gtccactgaa tacacagaca ctaggaatag gctgtcaaac 660
gacgcatgta gactcatttg agatcgttgc tgaaaatgaa aaactagcta tagtggatgt 720
cgttgatggc atagatcata aaataaattt aacaactact acatgcacaa ttcgaaattg 780
taagaagctg ggacctagag aaaacgtagc tgtaatacag gttggaggct ctgatatatt 840
agatattaca gcagatccaa cgaccaatcc acagactgag agaatgatga gagtgaattg 900
gaaaaagtgg tggcaagttt tttatacaat agttgattat attaatcaaa ttgtgcaagt 960
aatgtccaaa agatcacgat cgctaaacgc agcagccttc tattatagag tatagatata 1020
tcttagatta gaattgtatg atgtgaccgg atcc 1054
<210>3
<211>21
<212>DNA
<213>Artificial
<400>3
tggtattgaa tataccacaa t 21
<210>4
<211>21
<212>DNA
<213>Artificial
<400>4
aaggatggcc gacaggatca g 21
Claims (10)
1. A panda rotavirus CH-1 strain VP7 positive plasmid is characterized in that the VP7 positive plasmid is constructed by the VP7 gene and a PUC57 vector.
2. The VP7 positive plasmid of claim 1, wherein: and BamHI/SalI enzyme cutting sites are designed at two ends of the VP7 gene sequence.
3. The method of claim 2, comprising the steps of:
respectively treating the VP7 gene and the PUC57 vector by using BamHI/SalI enzyme;
carrying out nucleic acid electrophoresis analysis on the processed product and recovering 1048bp and 2697bp gene sequences;
and (3) connecting the 1048bp gene sequences with the 2697bp gene sequences to obtain the VP7 positive plasmid.
4. The method according to claim 3, wherein the treatment of the VP7 gene and the PUC57 vector with BamHI/SalI enzyme, respectively, comprises:
incubating reaction systems respectively containing the VP7 gene and the PUC57 vector at 37 ℃ for 3.5-5 h;
wherein the reaction system comprises 2ul BamHI/SalI enzyme, 10ul 10 × buffer, 10ul VP7 gene/PUC 57 vector and 33ul ddH2O。
5. The construction method according to claim 4, wherein the connecting 1048bp and 2697bp gene sequences comprises:
incubating the reaction system containing 1048bp and 2697bp gene sequences overnight at 4 ℃, and then incubating for 3.5-4.5 h at 16 ℃;
wherein, the reaction system comprises: 1ul T4DNA library, 1ul 10 XBuffer, 6ul VP7 gene and 2ul PUC57 vector.
6. An Escherichia coli strain containing the VP 7-positive plasmid according to any one of claims 1 to 2 or the VP 7-positive plasmid obtained by the construction method according to any one of claims 3 to 5.
7. The Escherichia coli strain according to claim 6, wherein the VP7 positive plasmid constructed by the VP7 positive plasmid according to any one of claims 1 to 2 or the construction method according to any one of claims 3 to 5 is transformed into Escherichia coli TOP10 competent cells.
8. The VP7 positive plasmid of any one of claims 1 to 2 or VP7 positive plasmid constructed by the construction method of any one of claims 3 to 5 is used as a positive control in PCR detection of panda rotavirus.
9. A PCR detection kit for detecting panda rotavirus, which is characterized by comprising a VP7 positive plasmid in any one of claims 1 to 2 or a VP7 positive plasmid constructed by the construction method in any one of claims 3 to 5.
10. A PCR detection kit for detecting panda rotavirus, which is characterized by comprising a standard substance, wherein the standard substance is VP7 positive plasmid extracted from the Escherichia coli strain of claim 6 or 7.
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