Protein standard substance of tomato spotted wilt virus and application of protein standard substance
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A standard substance and protein technology, applied in the field of protein standard substances, can solve the problems of lack of research products, and achieve the effects of high solubility, complete protein modification, and easy storage and transportation.
Pending Publication Date: 2021-04-09
昆明海关技术中心
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Domestic research and development of positive quality control for molecular biology detection of animal viruses, such as the construction of real-time quantitative PCR positive qua
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Embodiment 1
[0033] Embodiment one, the fusion of TSWV viral coat protein and replicase gene
[0034] The selected coat protein gene and the specific region of the replicase gene are amplified with specific primers,
[0035] RdRp-F: 5'-ATGAACATCCAGAAAATACAAA-3' and
[0036] RdRp-R:5'-GACTTCAGATTTGATCTTTTGAG-3';
[0037] CP-F: 5'-ATGTCTAAGGTTAAGCTCACTAAGGA-3' and
[0038] CP-R: 5'-ACAGACAAAACTTGCAGAACTTGCT-3' Obtain the replicase gene fragment and the full-length coat protein gene, press figure 2 The structure shown, the design of fusion protein primers,
[0039]RdRp-R: 5'-CTCGCTGCCGCTAGAACCACCAGAAGAACCACCAGAGACTTCAGATTTGATCTTTTGAG-3' and
[0040] CP-F: 5'-TCTGGTGGTTCTTCTGGTGGTTCTAGCGGCAGCGAGATGTCTAAGGTTAAGCTCACTAAGGA-3-Proceed through protein
[0041] Linker (SGGSSGGSSGSE) is connected, where the sequence protein Linker sequence is drawn.
[0042] And use RdRp RdRp-F: 5'-ATGGGTCATCATCACCATCACCATATGAACATCCAGAAAATACAAA-3' to introduce the His Tag sequence.
[0043] Using the same met...
Embodiment 2
[0044] Embodiment 2, the preparation of fusion protein
[0045] The expression vector was transferred into the 293 cell line, and after culture and fermentation, the cells were collected, broken up according to the method, and centrifuged to obtain the supernatant containing the expression product.
Embodiment 3
[0046] Embodiment three, the detection of standard substance
[0047] The standard substance obtained in Example 1 was measured by an ultraviolet spectrophotometer, and the virus value of the standard substance was converted according to the OD value, and the initial concentration of virus was measured to the power of 80,
[0048] It is proved that the standard substance can be used for quantitative analysis of TSWV.
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Abstract
The invention relates to a protein standard substance of tomato spotted wilt virus and an application of the protein standard substance. The protein standard substance is prepared by the following steps: firstly obtaining a whole-genome sequence for the tomato spotted wilt virus, and according to sequence comparison, obtaining a fusion protein expression vector containing a coat protein gene and a replicase gene, namely an XXX-CR vector; transferring the obtained eukaryotic expression vector of the coat protein gene and the replicase gene into a 293 cell strain, performing culturing and expressing, performing freezing and thawing at low temperature, performing centrifuging and performing purifying to obtain an expression product molecule; and performing DNase I digestion and purification, and performing quantifying to obtain the fusion expression protein standard substance of the coat protein and the replicase gene. The substance provided by the invention has the characteristics of good stability, no biological infectivity, ribonuclease resistance and the like, and is used as the standard substance in protein detection of the tomato spotted wilt virus.
Description
technical field [0001] This application relates to the field of protein standard substances, in particular, the present invention relates to a protein standard substance of tomato spotted wilt virus and its application. Background technique [0002] Tomato spotted wilt virus is a class of plant viruses worthy of attention. The number of virus types or isolates is increasing, and the range of hosts is also expanding. TSWV alone can infect 1090 kinds of plants, involving tomato, pepper, lettuce and other vegetable crops, peanuts, peas, tobacco and other important economic crops, as well as chrysanthemums and calla lilies. and many other flower crops. This genus is distributed in most parts of Asia and America. The harm caused is light and the yield is reduced, and the serious one is no harvest, and the huge economic loss caused has been listed as one of the ten most harmful plant viruses in the world. Tomato spotted wilt virus is a kind of virus that has increased the damag...
Claims
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