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Preparation method of a recombinant nuclear polyhedrosis virus capable of infecting tea geometrids

A karyotype polyhedron and recombinant virus technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, viruses/phages, etc., can solve the problem of high cost of EcobNPV, difficulty in large-scale rearing, and immature artificial rearing technology of tea inchworm and other problems, to achieve the effect of easy production control and low cost

Inactive Publication Date: 2011-11-30
溧阳市民生农业科技园有限公司 +1
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  • Abstract
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  • Application Information

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Problems solved by technology

[0005] Breeding EcobNPV with the above method has obvious shortcomings: the artificial breeding technology of tea geometrid is immature, and it is difficult to raise on a large scale, so the number of viruses propagated by artificial breeding of tea geometrid cannot meet the requirements of biological prevention of tea geometrid, and the cost of producing EcobNPV by this method is very high

Method used

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  • Preparation method of a recombinant nuclear polyhedrosis virus capable of infecting tea geometrids
  • Preparation method of a recombinant nuclear polyhedrosis virus capable of infecting tea geometrids
  • Preparation method of a recombinant nuclear polyhedrosis virus capable of infecting tea geometrids

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Embodiment 1

[0034] Embodiment 1: The construction of recombinant nuclear polyhedrosis virus capable of infecting tea geometrids includes the following steps,

[0035] 1. According to the complete genome sequence of EcobNPV registered in GenBank (accession number is DQ837165), primers were designed at both ends of the viral helicase gene:

[0036] SEQ ID No. 1: Eo-Helicase 1: GA ACTAGT ATGATTGGTGTAATCGT (underlined Speech I site),

[0037] SEQ ID No. 1: Eo-Helicase 2: CA CTGCAG ACTTTGCACGTAGTTTGTGTATATG (underlined Pst I site),

[0038] 2. Using EcobNPV DNA as a template, carry out PCR amplification with Eo-Helicase1 and Eo-Helicase1 primers. For the agarose gel electrophoresis pattern of the PCR product, see figure 1. Cloning the amplified EcobNPV envelope protein odv-e25, odv-e28 and helicase gene fragments (about 5.0 kb) into the pMD19-T (TAKARA company product) vector to obtain pMD19-helicase;

[0039] For the identification results of pMD19-helicase, see figure 2 . Fo...

Embodiment 2

[0050] Embodiment two: the application of the recombinant virus BmEoNPV that embodiment one obtains

[0051] The recombinant virus polyhedron that embodiment one obtains is prepared 10 8 The virus liquid of polyhedron / ml is sprayed on tea trees, and the 3-4 age tea geometrids caught in the field are stocked in tea trees sprayed with recombinant virus polyhedrons, and they feed naturally. After one week, the natural infection mortality of tea geometrids up to 63%.

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Abstract

The invention discloses a preparation method of recombinant nuclear polyhedrosis virus (NPV) which infects ectropis oblique. The preparation method comprises steps that: (1) recombinant vectors pFastBacDual-polh-helicase are prepared; (2) the recombinant vectors pFastBacDual-polh-helicase are transformed into E.coli DH10 BmBacmid competent cells; the cells are cultivated on a cultivating plate; positive plaques are obtained through blue-white selection; and recombinant BmBacmid-polh-helicase DNA is extracted from the positive plaques; (3) the ecombinant BmBacmid-polh-helicase DNA is transfected to domestic silkworm cultivated cells; cultivated cell supernatant is collected; and recombinant virus BmEoNPV is obtained; (4) the recombinant virus BmEoNPV is used for injection; and recombinant virus polyhedron is obtained. The recombinant nuclear polyhedrosis virus is sprayed on tea tree leaves, such that the ectropis oblique can be prevented from harming the tea trees. The preparation method also has advantages of low cost and controllable productions.

Description

technical field [0001] The invention relates to the construction of a recombinant virus in the field of genetic engineering, in particular to a method for genetically recombining the silkworm nuclear polyhedrosis virus to construct a genetic engineering virus. Background technique [0002] tea inchworm ( Ectropis oblique ), also known as arch worm, measuring worm, hanging silkworm, is the main pest of tea tree, and this worm belongs to Lepidoptera (Lepidoptera) geometridae (Geometridae). The larvae eat leaves, and when an outbreak occurs, the entire tea garden can be eaten into polished rods, resulting in a reduction in tea yield of more than 60%, which seriously affects the quality of finished tea. [0003] In the past, the basic methods of controlling tea inchworms were strengthening forecasting and agricultural control, manual hunting, and scientific use of chemical pesticides. However, the long-term use of chemical pesticides in large quantities has increased the resis...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/63C12N15/66C12N7/01A01N63/00A01P7/04
Inventor 杨益民贡成良潘中华曹广力薛仁宇刘卫星郭睿
Owner 溧阳市民生农业科技园有限公司
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