3). Gene expression technology
The recombinant vector in the recombinant engineering cell exists in two forms, one is in the cytoplasm in an independent recombinant vector, and in the cell division and proliferation, the vector is evenly distributed to the two cells after division, and there is a certain probability that the passage is gradually reduced, and finally the copy number of the recombinant vector is reduced, even the case of the recombinant vector is lost; the other is that the recombinant vector (including at least the gene of interest and the marker gene) is integrated into the chromosome of the host cell, and when the cell proliferates and divides the gene of interest and the marker gene are present on the chromosomes of the two daughter cells as the chromosome is replicated.
The recombinant engineered cells can realize the transcription and translation process of the target gene through the recombinant vector or the protein expression system of the cell itself, and express the target protein.
As a prokaryotic expression vector, recombinant Escherichia coli mainly induces protein expression through culture conditions, mainly including intracellular inclusion body expression, intracellular soluble expression and extracellular secretion expression. Yeast cells are primarily a means of inducing secretion of a protein of interest. Mammalian cells generally have inducible expression, transient expression and stable expression, such as promoters, enhancers, signal peptides, ribosome binding sites, transcription initiation signals, and transcription termination signals of the target gene in the recombinant vector or cell chromosome, translation initiation codon, translation stop codon, splicing signal, polyadenylation signal and other regulatory genes, as well as various enzyme genes, to achieve the transcription, translation and modification of the target gene, and finally it is expressed intracellularly and secreted extracellularly. For the stabilization of the protein of interest or for easy subsequent isolation screening, fusion protein genes are often constructed to express fusion proteins. As a recombinant protein drug, it is in principle required to finally separate the protein of interest in the fusion protein.
4). Genetic stability and expression stability
Genetic stability and expression stability are often determined using plasmid loss rate detection methods. In the case where the recombinant expression vector is independently present in the host cell, a pressurized method is generally employed to ensure the stable presence of the recombinant vector in the recombinant engineered cell, that is, in the presence of antibiotics, only the engineered cells which ensure a certain number of recombinant plasmids can grow normally. Engineering cells that lost the recombinant plasmid were destroyed. However, in the production process, the medium generally cannot contain antibiotics, so it is necessary to select those engineering cells which can be passaged in a non-resistant pressurized manner for a sufficient number of generations without losing the recombinant plasmid as a seed batch for production. For example, if an engineered bacteria needs to be passaged for 60 generations in the production process, we will culture it for 20 to 30 generations without antibiotics, and then spot the culture solution on a non-antibiotic solid medium plate for a total of 100. After being grown into suitable bacteria, they are inoculated on a plate containing antibiotic solid medium, and if 100 colonies are grown, the plasmid stability rate is 100%; repeating 4-6 times, that is, the engineering bacteria in the non-resistant medium is passaged for 90 to 120 generations. If the plasmid stability rate is not less than 95%, the strain can be used as a seed for production. Otherwise, it is necessary to re-screen and even re-cloning and transfecting the constructed engineering cells.
In the case of integration of the recombinant vector into the host cell chromosome, it is necessary to continuously increase the level of pressurization to screen for the engineering cells that integrate the highest target gene. For example, the genetic marker gene DHFR (dihydrofolate reductase) on a recombinant expression vector has the effect of resisting MTX (methotrexate), and when DHFR is integrated into the host cell chromosome along with the gene of interest, the project cells have the ability to grow and multiply in MTX-containing medium; the MTX concentration in the medium is gradually increased from 0.05 μmol/L to 5 μmol/L, and the DHFR gene copy number on the chromosome of the surviving engineered cells will only be resistant to 0.05. The engineering cells of μmol/L MTX are 100 times higher, that is, the copy number of the target gene on the chromosome of the engineering cell is increased by 100 times, and most of the cells with lower copy number are destroyed. The surviving engineered cells have a high expression level of the target protein, which ensures the expression of the target protein in the production process, and the target gene integrated into the chromosome is not easily lost, and MTX is not required to ensure its inheritance in the production process. stability.
(3). Other related technologies
1). Gene sequencing
The purpose of gene sequencing is to determine the ordering sequence of nucleotides in genes. Since 1975, three generations of sequencing technology have been developed.
The first-generation sequencing technology, also known as Sanger sequencing, adds a certain proportion of labeled dideoxynucleotides ddNTP (ddATP, ddCTP, ddGTP, ddTTP respectively) to the synthesis reaction system (containing polymerase, primers, DNA to be tested, and 4 kinds of dNTPs), since ddNTPs do not contain hydroxyl groups at both ends, the phosphodiester bond cannot be formed in DNA synthesis, thus interrupting the synthesis reaction, forming labeled DNA fragments of different lengths, by gel electrophoresis and autoradiography. The DNA sequence to be tested is determined based on the position of the electrophoresis band.
The second generation of sequencing technology is based on the needs of the "Human Genome Project". After continuous technology development and improvement, it is represented by Roche 454 technology, Illumina Solexa, Hiseq technology, ABI Solid technology and ThermoFisher IonTorrent technology. The core is to label four different nucleotide bases with different colors of fluorescence, mainly by the timing of the occurrence of bases. Compared with the first generation, the cost is reduced, the time is shortened, the throughput is increased, and the accuracy is improved, but the sequencing length is short.
The development idea of the third-generation sequencing technology is to maintain the speed and flux advantage of the second-generation technology and make up for the shortcomings of short reading. The main representative technologies are Oxford Nanopore's nanopore current detection technology, PacBio's SMRT technology, and Helicos' single-molecule fluorescence reversible termination technology. Compared with the previous two generations, the biggest feature is single-molecule sequencing, which does not require PCR to amplify DNA fragment.
Others have also summarized IonTorrent technology into third-generation sequencing technology, and called the nanopore sequencing as fourth-generation sequencing technology.
To be continued in Part Six…