Preparation of genetic engineering vaccine

Preparation of genetic engineering vaccine

1. Enzymes and carrier enzymes for the preparation of genetically engineered seedlings are indispensable reagents for genetic engineering. The tool enzymes used in genetic engineering are mainly restriction endonucleases and DNA modifying enzymes (ligases). Restriction endonucleases are referred to as restriction enzymes for short. Restriction enzymes are mainly enzymes that can specifically recognize 4 to 6 nucleotide sequences and have specific break positions or cut points, such as EcoRI, HindIII, PstI, BamHI, etc. DNA modification enzymes connect DNA fragments of target genes with carrier DNA molecules in vitro to form recombinant DNA molecules. Such enzymes mainly include T4 DNA ligase (separated from E. coli infected by T4 phage).

The gene carrier can carry foreign DNA fragments and enter the host cell for proliferation and expression. As a gene carrier, it should have the following characteristics: it can replicate itself in the host cell, even if there is a foreign DNA fragment covalently linked to it Affect its replication; should have a suitable restriction enzyme recognition site in order to connect with foreign DNA fragments; should have certain genetic markers to facilitate the selection of recombinants. Commonly used vectors are plasmids (such as E. coli plasmids pBR322, pSC101, Co1EI), bacteriophages (lambda phage and M13 phage), cohesive plasmids (constructed from plasmid DNA and the COS region of lambda phage, which not only has the Advantages, and can connect with large fragments of foreign DNA to form recombinant DNA molecules) and viruses (such as vaccinia virus, fowlpox virus, adenovirus, papovavirus, etc.).

2. The basic procedure generally includes the following five steps:

The first step is to isolate the target gene. There are two main methods for obtaining the target DNA fragment. One is to directly isolate it from the cell genome, and the other is to synthesize it manually.

The second step is to connect and recombine DNA fragments and vectors in vitro to form recombinant DNA molecules, which are mostly connected by ligase.

The third step is gene cloning, that is, introducing recombinant DNA molecules into suitable host cells (E. coli, yeast) for propagation. Depending on the vector used, transformation can be used (recombinant DNA molecules enter competent host cells in this way to obtain transformant colonies in the case of plasmids as vectors), transfection (recombination constituted by lambda phage as vectors) DNA molecules enter host cells in this way and can be transfected to get plaques), transduction (lambda phage DNA and foreign DNA composed of recombinant DNA molecules, and phage protein assembled into infectious phage particles, That is, the artificially packaged phage particles are introduced into the host cell), and recombinant DNA molecules are introduced into the host cell.

The fourth step is the screening and identification of target gene clones, that is, the cells carrying the target gene are isolated from a large number of cells carrying recombinant DNA molecules. Because not all cells can obtain recombinant DNA molecules, in order to obtain cells that have taken up the recombinant DNA molecules, they need to be screened to distinguish them from cells that have not taken up the recombinant DNA molecules and to be further identified. The methods for screening cells containing recombinant DNA molecules are generally based on the genetic markers and molecular characteristics of the carrier DNA and the target gene, combined with the genetic phenotype of the recipient cell. Since many plasmids have resistance markers for antibiotics and other drugs, on a selective medium containing a certain concentration of antibiotics, cells that have taken up the recombinant DNA molecules can be easily identified because cells that have also acquired antibiotic resistance . However, drug screening is only one aspect. According to it, it can only determine whether the plasmid vector has entered the recipient cell, and it cannot be determined whether the recipient cell has taken up the recombinant DNA molecule containing the target gene.

The identification of recombinant DNA molecules is usually carried out after extracting recombinant plasmid DNA, observing the size of the molecule by gel electrophoresis or electron microscopy, digesting with restriction enzymes, and observing the digestion pattern. The colony in situ hybridization method can also be used for identification, that is, the colony is transferred from the initially grown plate to the nitrocellulose filter, and the colony on the filter is lysed with alkali to free, denature and fix the DNA molecule on the filter Then, the DNA or RNA probe complementary to the target gene is hybridized with the isotope label, and finally the colonies are identified by autoradiography of the filter, and the colonies positive for autoradiography are selected from the originally grown plate.

The fifth step is gene expression, which refers to the transcription and translation of foreign DNA in the host cell during the mass reproduction of the host cell. The resulting product (protein) is preferably not decomposed inside the cell but secreted outside the cell . If the expression product is a small polypeptide or a protein that is extremely sensitive to bacterial proteases, it is usually rapidly degraded after it is formed. In order to ensure that the expression product of the foreign gene can be secreted outside the cell without being degraded, the foreign gene can usually be inserted between certain structural genes of the vector. In this case, the expression product is a fusion protein, and the fusion protein can resist internal The degradation of the source protease can be secreted out of the cell under the guidance of the cell signal peptide.