A new method for recombinant baculovirus-infected silkworm

The invention discloses a novel method for infecting silkworms with recombinant baculovirus. The method comprises the following steps: silkworm larvae are gathered into a cultivation frame; in a standard that each 100 silkworm larvae are ejected with 5ml of recombinant baculovirus liquid, the recombinant baculovirus liquid with 106pfu is ejected to the body surfaces of the silkworm larvas; and the silkworm larvae are not fed with mulberry leaves, and after most part of the baculovirus liquid is dried, the silkworm larvae are fed in an ordinary method till the silkworm larvae have the symptom of virus infection. The method disclosed by the invention has the advantages that a traditional inoculation method of percutaneous infection which is used directly is changed, the inoculation operation is simplified, the inoculation time is saved, and the working efficiency of an insect baculovirus expression system is improved.

The present invention relates to a recombinant virus-infected technology, particularly to a method for recombinant virus-infected silkworm.

BACKGROUND

The baculovirus expression vector system (BacuLovirus Expression Vector System, referred BEVS) is the use of insect carrying foreign gene recombinant baculovirus as vector in insects or insect cell culture expression of the recombinant protein produced by a production system. Because the system has an individual can take advantage of the insect cell culture or large-scale production of high expression of the advantages and become one of the most effective eukaryotic expression systems. Currently BEVS including Europe and the United States are widely clover guess looper nuclear polyhedrosis virus iAutographa californicanucleopolyhedrosis virus, referred AcNPV) and in China, Japan, the application of more of Bombyx mori nuclear polyhedrosis disease Orioles iBombyx mori nucleopolyhedrosis virus, Acronym BmNPV) two types of expression system (Smith et al, 1983;. Maeda et al, 1984,1985).. Silkworm BmNPV expression system can be used due to the amount of China's largest economic insect rearing silkworms as an expression vector, which has a low cost, suitable for large-scale production advantages, very with Chinese characteristics (吕鸿 Sound, 1998). However, current techniques are based on the infection of the recombinant virus injected directly into silkworm expression infection method, in actual operation due to the need to execute silkworm injection, it takes a long time, complicated operation, and because the injection process due silkworm parasite motility and other factors, the virus was out of the body, eventually leading to infection failed, so now the traditional method of percutaneous injection flaws obvious inefficiency. In order to improve the efficiency of a baculovirus expression system, it is necessary to develop a more efficient way of infection. Through trial and error, we found that the virus was applied to the surface silkworm larvae, the virus enters the parasites can be achieved, resulting in viral infections, and percutaneous injection to achieve the same effect infected O

SUMMARY

The present invention is to provide a new method for recombinant baculovirus by silkworm skin infections.

To achieve the above object, the present invention is used as follows:

The present invention is a recombinant baculovirus Bombyx mori infected silkworm percutaneous method is: will focus on raising silkworm box; to 100 5ml injection of recombinant Bombyx mori silkworm baculovirus standard solution containing IO6 Pfu recombinant baculovirus Bombyx mori liquid jet in silkworm larvae surface; after injection not fed mulberry leaves within I hour, until most of the virus was dried silkworm larvae reared in a conventional manner, until the emergence of symptoms of viral infection silkworm larvae.

Further, the present invention is the 3rd instar silkworm larvae or fourth instar.

Compared with the prior art, the present invention has the advantages that:

Results of the method (I) of the present invention shows that by the recombinant baculovirus Bombyx mori silkworm larvae liquid spray to the surface, but also make the virus enters the silkworm; because of silkworm and other insects, as there is an open circulatory system Once the virus enters the bloodstream, the virus can quickly infect, and to the proliferation of the surrounding tissue, and ultimately systemic infection.

(2) silkworm - baculovirus expression system is likely to become China's biotechnology industry a powerful tool, the present invention solves the recombinant virus infection complicated operation, low efficiency problem, simplified method for viral infections greatly improve the efficiency of the baculovirus expression system. Brief Description

(I) Figure I is a blood sample silkworm larvae inverted fluorescence microscope photographs, which Figure I (a) None of the silkworm larvae infected blood samples; Figure I (b) to be reorganized silkworm rod Blood samples silkworm larvae-like virus infection, and the emergence of green fluorescence.

(2) Figure 2 is a non-infected silkworm larvae with the recombinant baculovirus-infected silkworm silkworm larvae symptom control chart, which Figure 2 (a) for the non-infected silkworm larvae; Figure 2 (b) to be reorganized silkworm Bombyx mori baculovirus-infected larvae.

DETAILED DESCRIPTION

The following provides further details on the method of the invention:

I. Materials: Recombinant baculovirus using silkworm has a very mature technology to build, specific methods can be found in papers published at home and abroad, such as in 2006, published in "Science of Sericulture" and "the use of bacterial transposon construct suitable for silkworm Bac-to-Bac rapid gene expression system "article 32 (2): 183_188. With the above silkworm Bac-to-Bac expression of the reporter gene technology to build a green fluorescent protein EGFP recombinant baculovirus Bombyx mori, the use of the virus, using the method of the present invention percutaneously infected silkworm larvae.

In the following examples, the use of silkworm hybrids, varieties name Qiufeng X white, mulberry silkworm breeding, rearing temperature of 23~25 ° C.

In the following examples, the method of percutaneous recombinant baculovirus infected silkworm Bombyx mori follows:

The 3rd instar silkworm larvae or 4 concentrate on keeping the box; injection 5ml to 100 silkworm virus solution as the standard, with a small sprayer containing IO6 pfu (viral infectious units) of recombinant baculovirus Bombyx mori silkworm larvae liquid jet in the body table, and I is not fed mulberry within hours after injection; after most or all of the virus after the liquid drying, silkworm breeding by a conventional method, until the silkworm larvae appears as shown in Figure 2 (b) of the symptoms of viral infection.

Determination of effect of infection: in recombinant baculovirus Bombyx mori silkworm larvae liquid spray to the surface after 72-96 hours, take a small amount of blood silkworm larvae placed on a slide, see if you have a viral infection in the inverted fluorescence microscope green fluorescence produced [Figure I (b) shown], if there is, then that is a recombinant Bombyx mori silkworm baculovirus infection. Liquid injection of recombinant baculovirus in silkworm silkworm larvae in 120 hours or so after the body surface, further observation of the onset of symptoms of silkworm larvae.

Analysis: In 96 hours the recombinant baculovirus Bombyx mori silkworm larvae liquid spray to the surface after about, when observed under a fluorescence inverted microscope silkworm larvae of blood, you can find a clear green fluorescence [see Figure I (b)]; blood samples without silkworm larvae infected with the virus will not produce sudden light [see Figure I (a)].

In 120 hours of recombinant baculovirus Bombyx mori silkworm larvae liquid spray to the surface after about silkworm larvae can be found in the typical symptoms of viral infection, such as loss of appetite, shiny surface, somites swelling, manic crawling etc. [comparison chart 2 (a) and Figure 2 (b)], and further illustrate silkworm larvae have been infected, recombinant proteins - green fluorescent protein was expressed.