DE19509169C2 - Primer and alpha-fetoprotein produced with the aid of such a primer - Google Patents

Description

The invention relates to a primer, in particular used for the genetic engineering of alpha-fetoprotein is cash.

From US 5 206 153 (Chemical Abstracts, Volume 119, 1993, Page 298) is a process for manufacturing human Lich alpha fetoprotein known in which by means of trans genetic yeast cells with the signal peptite of alpha-feto protein-bound human alpha feto protein is produced.

Primers are short single-stranded nucleotide sequences that are called Starting point of replication for DNA synthesis. You leave are involved in cDNA synthesis, among others, in which eukaryotic, polyadenylated mRNA is assumed. Dar In addition, primers in PCR (polymerase chain cracks tion) of importance.

The choice of a suitable primer is often decisive for the course of the polymerase reaction and for the formation of the desired polynucleotide strand. For example, wise from a mixture of different mRNA or DNA frag select only certain fragments for cloning and not have to create an extensive gene bank attempts are made to amplify only the DNA of interest ren.

The object of the present invention is therefore a sol Chen primer to provide a suitable replicate starting point for DNA synthesis. In particular is it object of the present invention, by using a such primers the genetic engineering of alpha-feto favor protein.  

According to the invention, the task is solved by a primer for the synthesis of a polynucleotide strand, selected from the group of oligo nucleotides

• a) 5 ′ -CCCGGATCCAACAAAATAACTAGCAACCATGA
• b) 5 ′ -CGGGTCCCAAAATTTTTCATTA
• c) 5 ′ -CCCAAGCTTCGTTTTGTCTTCTCTTCCCC
• d) 5 ′ -TTTTGGGACCCGAACTTTCCA

where A = adenine, T = thymine, G = guanine and C = cytosine points.

According to a preferred embodiment of the invention used in a mixture of oligo nucleotides a) to d), where in particular the primer pairs a) + b) and c) + d) ver be applied. This allows a significant improvement cDNA synthesis and PCR, associated with a specific DNA amplification.

The primers according to the invention are suitable for cloning DNA. Here, the DNA can basically be any nucleotide own a consequence. Good results have been achieved especially with the Cloning of DNA obtained, the alpha-fetoprotein (AFP) ko dated.

The AFP-encoding DNA here has a sequence according to FIG. 1 or 2, and modifications of the DNA, for example by deletion, substitution, addition, inversion or inversion of nucleotides, are also possible. In Fig. 1, the AFP sequence and in Fig. 2, the operon sequence shown.

The present invention therefore also relates to a process for the production of a protein using a primer according to the invention or a mixture of these primers and the protein produced in this way. This pro tein can in particular be alpha-fetoprotein with an amino acid sequence according to FIG. 3, which makes it possible to replace the naturally obtained alpha-fetoprotein, so that, above all, the risk of pathogenic impurities in the product is avoided. Due to its purity, the genetically engineered alpha-fetoprotein can be used, among other things, for immunodiagnostics.

The present invention is explained in more detail below with the aid of the cloning scheme shown in FIG. 4.

The example shown is based on genomic DNA, which carries the gene for human alpha-fetoprotein. The The structure of this gene is typical for eukaryotes divided into exons and introns, so that after splicing the primary transcription product is the polyadenylated mRNA arises. This mRNA can be reverse transcribed tion in turn generate DNA, the so-called cDNA. This cDNA possesses complete genomic DNA expressible coding sequences, which are preferably the Cloning are suitable.

The general problem with producing the cDNA is that when using non-specific oligo-d (T) primers very ver different clones are available, for example by means of hybridization or immunodetection must be chosen that contain the gene of interest.

To avoid this effort, according to the present Invention both for the preparation of the cDNA as well used for the application of the PCR specific primer from the group of oligo nucleotides

• a) 5′-CCCGGATCCAACAAAATAACTAGCAACCATGA
• b) 5′-CGGGTCCCAAAATTTTTCATTA
• c) 5′-CCCAAGCTTCGTTTTGTCTTCTCTTCCCC
• d) 5′-TTTTGGGACCCGAACTTTCCA

is selected.

Here it is advantageous for the replication of the nucleo tid strands to use primer pairs, preferably assign the primer pairs a) + b) and c) + d). As a result of the DNA Polymerization thus results in two overlapping ones DNA fragments used in the next step for insertion in a suitable vector can be used. Can be used as a cloning vector for example, the plasmid pMGCAFP serve.

The expression of the AFP gene is then followed by an over delivery of the gene into a host cell, especially a human cell, a chicken or cattle cell. One serves the transduction, the recombined cDNA initially integrated into a virus genome. Preferably serves the vaccinia virus with which the host cell connects can finally be infected. The host cell then expresses the foreign AFP gene and changes the AFP formed into that issuing medium.

When using mammalian cells as host cells, one could AFP concentration in the culture medium of 6% based on the Ge velvet proteins are detected. This corresponds to a con concentration of 1000 ME / ml. By usual cleaning procedures, e.g. B. by dialysis, the percentage of AFP further increase based on total proteins. Finally the AFP can also be full using affinity chromatography clean constantly.

Claims (7)

1. Primer for the synthesis of a polynucleotide strand selected from the group of oligo-nucleotides

• a) 5′-CCCGGATCCAACAAAATAACTAGCAACCATGA
• b) 5′-CGGGTCCCAAAATTTTTCATTA
• c) 5′-CCCAAGCTTCGTTTTGTCTTCTCTTCCCC
• d) 5′-TTTTGGGACCCGAACTTTCCA

where A = adenine, T = thymine, G = guanine and C = cytosine means. 2. Mixture of primers from claim 1. 3. Use of a primer or a mixture of primers according to claim 1 or 2 for cloning DNA. 4. Use according to claim 3, characterized in that the DNA has a sequence according to FIG. 1 or 2, wherein a modification of the DNA is possible in particular by deletion, substitution, addition, insertion or inversion of nucleotides, wherein Fig. 1 or 2 is part of this claim. 5. Process for the production of a protein, characterized by the following steps:

• Obtaining mRNA as a transcription product of the gene coding for the protein
• Conversion of the mRNA by reverse transcriptase into cDNA with the possibility of subsequent amplification using a primer or a mixture of primers according to claim 1 or 2
• - Cloning of the cDNA using suitable vectors
• - Expressing the protein coding gene in a mammalian cell.

6. The method according to claim 5, characterized in that the protein is alpha-fetoprotein.