Recombinant eggs and gene cloning and expression vectors based on avian adenoviruses

An adenovirus, avian technology, applied in the field of molecular biology, can solve problems such as expensive, difficult and unpredictable basic manufacturing process

Inactive Publication Date: 2003-04-02
坎默根公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using bacterial, yeast or insect cell culture systems as the basis for manufacturing processes remains difficult, unpredictable and expensive

Method used

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  • Recombinant eggs and gene cloning and expression vectors based on avian adenoviruses
  • Recombinant eggs and gene cloning and expression vectors based on avian adenoviruses
  • Recombinant eggs and gene cloning and expression vectors based on avian adenoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Using the results of Swedish researchers describing the homology between the hexon genes of human Ad2 and Ad CELO (J.Virology-1982, v.42, N1, 306-310), we synthesized some primers, which It is the unique characteristic sequence of human Ad2 hexon gene. Primer specificity was determined by sequencing cloned fragments from Ad CELO and human Ad5. One of the primers was used to synthesize an Ad CELO fragment that hybridized to the human Ad2 hexon gene. The reverse primer is from human Ad2 genome position: 21164-21185bp

[0062]5'AGGAACCAGTCTTTGGTCATGT-3' SEQ ID NO: 27

[0063] This primer has been used for cDNA synthesis of the Ad CELO hexon gene. First-strand cDNA synthesis was performed with AMV reverse transcriptase. For second strand synthesis RNase H, DNA-pol 1 and T4 DNA-pol were used. The double-stranded cDNA (approximately 2500-3500 nucleotide base pairs) was washed from the agarose gel and cloned into pBluescript II SK(+) vector. Clones were selected by molec...

Embodiment 2

[0065] This example describes the generation of the plasmid pSKII-DC5'-MLP-2bLd, which is depicted in Figure 1 . Plasmid pSKII-DC5'-MLP-2bld was prepared by directional cloning in 3 sequential steps.

[0066] 1. Cloning of the 5' end of the FAV1 genome

[0067] First, by using oligonucleotide primers and polymerase chain reaction (PCR), up to 538 base pairs (bp) of the left end of the Ad CELO genome (located between 0 and 538 bp DNA sequence of the avian adenovirus CELO genome Between) were amplified and isolated. The primers used were:

[0068] 5'-CAAGTGGTACCGGCCAAATTGGCCGATGATGTATAATAACCTCA-3' [SEQ ID

[0069] NO: 1]

[0070] 5'-CAACCAAGCTTCTCTTCCGAAGTCATCTG-3' [SEQ ID NO: 2]

[0071] The amplified sequence of 538bp is shown in Table 1 (SEQ ID NO: 21)

[0072] The amplified DNA contains the essential origin (ori) and packaging sequence (pkg). This kPCR fragment (KpnI-ori / pkg-HindIII) was digested with HindIII only and inserted into the pSKII vector, which was digested...

Embodiment 3

[0089] This example describes the generation of plasmid pSKII-DC5'-MLP-2BLD-RG-p(A), which is depicted in FIG. 2 . Plasmid pSKII-DC5'-MLP-2BLD-RG-p(A) was prepared in three consecutive steps by directional cloning. First, using oligonucleotide primers, reverse transcriptase (Amersham) and PCR, the glycoprotein gene of the virus was prepared from the RNA of the rabies virus vaccine strain Vnukovo-32, up to 1640bp of the rabies virus DNA sequence was amplified and separate. The primers used were:

[0090] 5'-GGATCCAGGAAAGATGGTTCCTCAGGCTCTCCTGTTTG-3' [SEQ ID NO: 9]

[0091]5'-GCTGCAGCAAGGGGAGGTGATCTTCAGACTTGGATCGT-3' [SEQ ID NO: 10]

[0092] The amplified DNA contains the glycoprotein gene of the rabies virus vaccine strain Vnukovo-32 sequence (RG). The pSKII vector was subsequently opened with BamHI-PstI, and the PCR fragment BamHI-RG-PstI was inserted therein. In the second step, using oligonucleotide primers and polymerase chain reaction (PCR), up to 240 base pairs (bp)...

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Abstract

We describe a new technology for producing eggs containing recombinant proteins based on the novel use of avian adenoviruses (AAV). Adenovirus infected embryonated eggs are used as the host organism. The proteins synthesized are isolated in high amounts per egg and purified excluding time-consuming steps of purification. As far as is known, the mechanisms of translation and post-translational modification of proteins in avian species fully resembles those of other higher eukaryotes such as humans, veterinary animals, and plants. In contrast with bacterial, yeast and plant host system s for producing recombinant proteins, avian embryos allow isolation of protein s in biologically active form free of endotoxins. The combination of high productivity and possibility of obtaining proteins in native form makes the system a unique choice for biotechnology, much more favorable than other hosts currently being used for this purpose.

Description

field of invention [0001] The present invention belongs to the field of molecular biology. The present invention particularly relates to eggs containing recombinant proteins and / or recombinant DNA and vectors and genes for the production of recombinant proteins in eggs. Background of the invention [0002] Transferring genetic information into marketable products is an emerging paradigm in the pharmaceutical industry. The market for biological products is expected to reach $150 billion by 2000, driven in large part by genomics and new drug discovery. This growth reflects the need for the molecular characterization of newly discovered genes whose functions may provide treatments for diseases. Once these genes are identified, larger quantities of production are often required for research and clinical trials that can lead to marketable products. There are few recombinant protein production technologies that can be used to produce products of clinical quality. Using bacteri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/235A61P31/20C07H21/02C12N5/00C12N5/02C12N7/00
CPCC12N15/86C12N2710/10243
Inventor V·I·格拉布科E·R·布莱登
Owner 坎默根公司
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