Recombinant chicken Marek's disease virus transfer vector and application thereof

A technology for Marek's disease and transfer vector, applied in the field of genetic engineering, can solve the problems of inability to overcome the interference of maternal antibodies and cannot be used for vaccinated birds, etc., to increase the immunization coverage, simplify the immunization procedure and reduce the cost. Effect

Inactive Publication Date: 2006-04-26
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, one of the most important disadvantages of poxvirus as a carrier is that it can cause carrier effects, so poxvirus vector vaccines cannot be used in poultry that has been vaccina

Method used

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  • Recombinant chicken Marek's disease virus transfer vector and application thereof
  • Recombinant chicken Marek's disease virus transfer vector and application thereof
  • Recombinant chicken Marek's disease virus transfer vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Construction of transfer vector

[0071] 1.1 Extraction of pcDNA4.0 / His / LacZ and pUS2 plasmids

[0072] Streak the Escherichia coli JM109 Amp / 2xYT solid medium plate with plasmids pcDNA4.0 / His / LacZ and pUS2, culture at 37°C for 20 hours, pick a single white colony and inoculate it in Amp / 2xYT liquid medium with shaking at 37°C Cultivate for 16 hours, and then proceed according to the operating instructions of the plasmid extraction kit of Biotech Bioengineering Company:

[0073] Collect 1.5~3mL of the bacterial liquid precipitate into a 1.5mL Eppendorf centrifuge tube, add 100μL of solution I, shake until completely suspended;

[0074] Add 150 μL of solution II, and immediately invert the centrifuge tube gently several times to fully lyse the bacteria, and the lysed bacteria become clear. Then place the centrifuge tube on ice for 2 min;

[0075] Add 150 μL of solution III, immediately invert the centrifuge tube gently several times, and place it at room tem...

Embodiment 2

[0127] Example 2. Application of Transfer Vector in Construction and Screening of Recombinant Chicken Marek's Disease Virus

[0128] 2.1 Construction and screening of recombinant chicken Marek's disease virus

[0129] Prepare chicken embryo fibroblasts (CEF) according to conventional methods, after it grows into 80% monolayer, wash 2 times with the diluent of MDV, then inoculate MDVCVI988 / Rispens vaccine virus, the inoculation dose is 50PFU per hole in 24 well plates (diluted with MDV diluent), after acting for 4 hours at 37°C, wash twice with MEM without antibiotics, according to Lipofectamine TM 2000 instructions, transfect the cells with the transfer vector plasmid, after 2 hours of action, pour off the transfection solution, add MEM culture solution containing 5% bovine serum at 37°C CO 2 After culturing in the incubator for 4-5 days, when typical cell lesions appear, add nutrient solution containing X-gal (200 μg / ml) to observe blue plaques, digest the infected CEF cells...

Embodiment 3

[0132] Example 3. Construction and Application of Recombinant Marek's Disease Virus Expressing Infectious Bursal Virus (IBDV) VP2 Gene

[0133] 3.1 Construction and screening of recombinant Marek's disease virus expressing infectious bursal virus (IBDV) VP2 gene

[0134] The 1.35kb IBDV VP2 gene was inserted into the pUS2-LacZ vector through the EcoR I and Xhol I sites to obtain the recombinant plasmid pUS2-LacZ-VP2, and the transfer plasmid pUS2-LacZ-VP2 was transfected with liposome-coated MDVCVI988 / For the CEF cells infected by the Rispens strain, according to the method in 2.1, the substrate X-gal was added 4 to 5 days after transfection, and the appearance of blue spots was observed, (such as Image 6 ) Screening of recombinant MDV by coeruleus purification.

[0135] 3.2 Identification of recombinant virus

[0136] The DNA of the obtained stable recombinant virus is extracted and purified, and a PCR product with a size of about 1.35 kb is amplified with specific primer...

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Abstract

The present invention relates to recombinant chicken Marek's disease virus transfer vector and its application. The transfer vector pUS2-LacZ features that the recombinant plasmid pUS2 has US2 gene segment substituted with CMZ immediate early promoter, LacZ gene, transcription termination signal SV40polyA, ampicillin and neomycin marked gene and one polyclonal site gene. The transfer vector may be homogeneously recombined with MDV vaccine virus to obtain recombinant virus MDV of LacZ marked gene, and has polyclonal site capable of inserting foreign virus gene to obtain recombinant plasmid. Through further homogeneous recombination, purification and screening, recombinant virus is obtained, and the recombinant virus may be prepared into vaccine with effect of resisting several kinds of virus.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a virus transfer vector and its application. technical background [0002] Recombinant live vector vaccine refers to the use of genetic engineering technology to transfer the protective antigen gene (target gene) into the corresponding carrier, and express the protective antigen protein after recombination with the virus gene. This vaccine can be passed once Immunization prevents a variety of diseases, significantly reduces the cost of vaccines, simplifies immunization procedures, and increases immunization coverage. At the same time, the protective antigen in this vaccine is single, which is conducive to the establishment of immune monitoring methods. Therefore, live vaccine viruses are used as carriers to study recombination Live vector vaccines are an important direction for future vaccines. [0003] With the development of molecular biology, the molecular sites and structu...

Claims

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Application Information

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IPC IPC(8): C12N15/63A61K39/12C12N7/01C12R1/93
Inventor 李永清周雪媚张培君
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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