This paper mainly introduces the concept of genetic engineering, the general process of drug development using genetic engineering technology and genetically engineered drugs, and discusses the development direction of drug development research using genetic engineering technology in the future.

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1 Overview of genetic engineering

The so-called genetic engineering refers to the insertion of nucleic acid molecules into viruses, plasmids or other vector molecules in vitro to form a new combination of genetic materials, which can be added to host cells without such molecules before, so that they can reproduce continuously and stably.

The first important feature of genetic engineering is that it can cross the barrier of natural species, that is, it can put the gene of any organism into the cells of a new host organism that has nothing to do with it. This shows that it is possible for people to create new species that do not exist in nature according to their own subjective wishes. The second feature is that it emphasizes the amplification of a small DNA fragment in a new host cell, so that a large and purified DNA fragment can be prepared, thus broadening the field of molecular biology and making it have great application in the field of biopharmaceuticals.

Since the advent of genetic engineering in the early 1970s, amazing achievements have been made in both basic theoretical research and practical application. The determination and analysis of the whole genome nucleotide sequence is an excellent example of genetic engineering technology promoting basic biological research. 2001February 12, scientists from six countries and the international human genome published the human genome map and preliminary analysis results, providing people with about 3,000 medicinal genes, which will promote the rapid development of gene pharmaceutical industry. Due to the development of gene cloning technology, genetic engineering technology has played an important role in industrial production, especially in pharmaceutical production. In the past, people used microorganisms to produce useful products, such as penicillin produced by Penicillium and streptomycin produced by Streptomyces. However, separating and purifying these drugs from these organisms is not only expensive, but also technically difficult. Nowadays, a large number of useful drugs can be easily extracted by cloning and transferring genes encoding these drugs into suitable organisms for effective expression.

2 General process of drug development by genetic engineering technology

The development of a drug by genetic engineering technology generally goes through the following steps: ① obtaining the target gene fragment: DNA fragment with known nucleotide sequence can be synthesized by chemical synthesis; It can also be extracted and separated from biological tissues and cells. Eukaryotes need to establish a cDNA library. ② After the obtained target gene fragment is amplified and connected with a suitable vector, it is introduced into a suitable expression system. ③ Under suitable culture conditions, the target gene can express a large number of target drugs in the expression system. ④ Extracting, separating and purifying the target drug, and then preparing the corresponding preparation.

Most of the above methods use microorganisms or tissue cells as expression systems, and produce drugs through microbial fermentation or tissue cell culture. In recent years, the "biopharmaceutical factory" that produces drugs through transgenic animals has become the most active field of transgenic animal research and the most attractive industry of genetic engineering pharmacy. Transgenic animal drugs have the advantages of low production cost, short investment cycle, high expression, complete consistency with natural products, easy separation and purification. It is especially suitable for some blood factors with large dosage and complex structure, such as human hemoglobin (Hb), human albumin (HSA) and protein C(Protein C). Edinburgh Pharmaceutical Company in the United Kingdom produced α 1- antitrypsin (α 1-AAT) through transgenic sheep to treat emphysema, and produced 16g AAT per liter of goat milk, accounting for 30% of the protein content of milk. It is estimated that each lactating ewe can produce 70 grams of AAT. In addition, transgenic plant drugs are safer than transgenic animal drugs, because the latter may pollute human pathogens. At present, many transgenic plant drugs have been developed, such as enkephalin, interferon-α and human serum protein, as well as the two most expensive drugs, namely glucocerebrosidase and granulocyte-macrophage colony factor.

Three kinds of genetically engineered drugs

Since the late 1970s, genetically engineered drugs have developed rapidly. 1978 The human brain hormone and human insulin expressed by synthetic genes were produced by E.coli for the first time. 1980, the US Supreme Court ruled that microbial genetic engineering can be patented. 1982, the first drug insulin produced by genetically engineered bacteria was approved for use in the United States and Britain. Since it was approved for use, various genetic engineering drugs have mushroomed. China's medical technology research and development and industrialization have also made great progress.

The traditional production of (1) antibiotics is mainly obtained by chemical synthesis or microbial fermentation. In the production process, the expression level of the strain is low, the production cost is high, and drug-resistant flora is easy to produce in the use process. Genetic engineering technology can be used to genetically modify the production strains to obtain strains with high expression level and strong product purpose, such as penicillin phthalamidase produced by Escherichia coli. A German research team used genetic engineering to enhance the penicillin amidase activity of Escherichia coli. The enzyme activity of the strain formed by cloning the plasmid of Escherichia coli gene PBR322 was 50 times higher than that of the original strain, thus improving the production capacity of 6APA. Wang Yiguang in China transformed spiramycin-producing bacteria by gene recombination technology, which enhanced the expression of propionyltransferase gene in spiramycin-producing bacteria and increased the yield of propionyl spiramycin.

(2) There are a series of active peptides in human body, such as hormones, with low content but high physiological activity, which play an important role in regulating human metabolism. These substances can be used as drugs to treat diseases caused by imbalance of such substances in clinic. The preparations of these drugs mostly come from various animal organs, and the production method is complicated and the cost is high. Individual products must be extracted from animal carcasses, and large-scale industrial production cannot be carried out. Since the emergence of genetic engineering technology, it can be produced from micro-vividness through gene recombination technology, which is one of the greatest achievements of genetic engineering technology. The following are two typical drugs of this type.

Insulin: 1978, Genentech developed the production of human insulin by using Escherichia coli by Goeddel and other scholars using gene recombination technology. With the continuous development of genetic engineering technology, the process and technology of producing insulin have been continuously improved, which has completely replaced the products extracted from animal organs in clinic. At present, transgenic sheep in Xinjiang have been able to successfully express human proinsulin, which opens up a new way for insulin production.

Auxin: Human auxin is clinically used to treat dwarfism and muscular dystrophy. The traditional manufacturing method is to extract from human pituitary gland, and the source of raw materials is difficult, so the output is greatly limited. Only 1% of dwarfism patients in the world can be treated, because auxin is extremely expensive, up to $5,000 per gram. 1979, Genentech was first developed and produced human auxin by Goeddel and other scholars using Escherichia coli. In recent years, yeast has been developed to produce auxin, and the yield can reach1.4×106 ~ 4.7×106 molecule/cell. At present, China's genetically engineered human auxin has been successfully developed and put into the market for clinical use.

In addition to the above drugs, there are nerve growth factor (PDGH) produced by genetic engineering technology, human basal fibroblast growth factor and chorionic gonadotropin.

(3) Genetic engineering technology of cellular immunomodulatory factors has been widely used in cellular immunomodulatory factors, such as anti-tumor and immunomodulation. In recent years, due to the progress of gene recombination and cell fusion, as well as the improvement of high-pressure liquid chromatography, amino acid sequence cleavage device and protein refined analysis technology, the research and development of some substances regulating cellular immune activity have developed rapidly, such as interferon (INF), interleukin (IL), colony stimulating factor (CSF) and tumor necrosis factor (TNF).

Interferon is a product with extensive research, mature technology and early industrialization. The first generation of interferon was extracted from blood. According to K Canted, Finland, the purity of interferon below 65,438+0% is less than 65,438+000 mg after treating 23,000 L of blood, so the yield is very low. Moreover, because the quality of blood source cannot be guaranteed, it may cause the spread of blood-borne infectious diseases. The second generation interferon is produced by genetic engineering technology. Its production level can reach 250,000 molecules/cell, and each liter can contain 250 million units. The cost is obviously reduced, and the product purity is very high, and the content can reach more than 90%. At present, there are three commercial genetic engineering interferons: α, β and γ, and the production technology is constantly improving. Russian scientists have constructed an expression system with Pseudomonas as the vector to produce genetically engineered interferon. Compared with the traditional Escherichia coli expression system, the culture cycle is shorter and the cells are easy to be broken and extracted. With the continuous development of gene recombination technology, some researchers have modified interferon gene and constructed targeted interferon gene and expression vector. Xia et al. used restriction endonuclease to cut the target gene from the plasmid containing human anti-HBV S antigen single chain antibody and human interferon α, respectively, and connected it to pET22b plasmid to construct a single chain antibody targeting interferon expression vector, which was successfully expressed in E.coli..

(4) Vaccine Traditional vaccines are attenuated or inactivated substances of pathogenic microorganisms, but these vaccines are not ideal, and may undergo reversion mutation to restore toxicity; Or an epidemic caused by improper inactivation. The new vaccine produced by genetic engineering technology can overcome the shortcomings of high price and poor safety of traditional vaccines, and can provide effective treatment for some special diseases such as AIDS, for which there is no effective vaccine at present.

The first commercial genetically engineered vaccine was against human hepatitis B virus (HBV). About 65,438+00% of the population in China have been invaded by HBV, and HBV infection is usually closely related to special liver cancer (HCC), and about 300,000 patients worldwide die of HCC every year. HBV has high host specificity and can only infect humans and chimpanzees, which means that only a limited number of viruses can be obtained from hepatitis patients as vaccines, and vaccines extracted from patients' blood may also be infected with AIDS. The anti-HBV vaccine produced by genetic engineering technology overcomes the shortcomings of traditional vaccines, with high quality, good safety and small dosage. The general dosage is below 10mg, and it is inoculated three times, which is one thousandth of the dosage of ordinary drugs. 1982, P. valen zuela and others cloned a fragment of S gene (HBV surface antigen gene) on the vector. As a result, HBV surface antigen (HbsAg) particles were synthesized in yeast with a yield of 25 μ g/L. Now the yeast expression system has been able to produce recombinant hepatitis vaccine for human use on a large scale.

About 20 years ago, it was found that "naked" DNA injected into the body could induce immune response. Scientists have done a lot of research and developed a new type of nucleic acid vaccine. The so-called nucleic acid vaccine refers to the direct transfer of exogenous genes (DNA or RNA) encoding antigen proteins into animals, and the synthesis of antigen proteins through the host expression system, thus inducing the host to produce immune responses to the antigen proteins, so as to achieve the purpose of preventing and treating diseases. A variety of nucleic acid vaccines have been developed, such as influenza nucleic acid vaccine, AIDS vaccine, rabies vaccine, tuberculosis vaccine, hepatitis B vaccine and hepatitis E vaccine.

(5) Gene therapy of gene therapy products started on 1990. In 1993, FDA defines human gene therapy as "medical treatment based on genetic material changes of living cells, which can be performed outside the living body and then applied to the human body, or directly performed in the human body". Therefore, there are two ways of gene therapy, namely, indirect in vivo method and in vivo method. Indirect in vivo method is mainly to screen cells that can express foreign genes through gene transfer in vitro, and then transfer them into the body; In vivo method is to directly change and repair genetic material in vivo. With the development of molecular biology and gene recombination technology, the method of obtaining the target gene has become mature, but the transfer and delivery system, expression regulation, effectiveness and safety of the target gene still need further study and confirmation. At present, gene transfer systems are divided into two categories: one is virus-mediated gene transfer system, which mainly includes retrovirus (Rt), adenovirus (Ad), herpes virus (HSV) and adenovirus-associated virus (AAV) vectors. Nnldini et al. developed a recombinant Rt vector based on HIV, which can infect a variety of non-splinter cell without helper cells, while retaining the characteristics of integration on the host chromosome. The first gene therapy vector in the world is Rt vector, which is used to treat severe combined immunodeficiency (ADA-SCID) caused by adenylate decarboxylase deficiency. The other is non-virus-mediated gene transfer system, including liposome, molecular coupling vector, gene gun and naked DNA.

In addition, antisense nucleotide technology is also used in gene therapy, especially for hepatitis B virus, including antisense DNA, antisense RNA and ribozyme RNA. 200 1, Robaczewska et al. selectively inhibited the replication and expression of HBV in the liver of Beijing duck by intravenous administration of antisense DNA for the first time, which proved the effectiveness of antisense DNA in animal experiments. Viagene Company of the United States has developed an "HIV immune preparation", which is a combination product of mouse retrovirus, core protein coding gene sequence and HIV surface antigen RNA, and it has been confirmed in mouse and primate experiments that the drug can induce strong HIV-specific killer cells.

4 conclusion

Genetic engineering technology has made fundamental changes in drug development. The traditional way of drug development is to randomly select the effective components as new drugs from a large number of chemical synthetic substances and microbial metabolites. Using genetic engineering technology to develop new drugs is to find the effective components and their coding genes that can be used for therapeutic purposes through the study of pathogenic mechanism, and then transfer them into appropriate vectors through gene recombination, so that the effective components can be expressed in large quantities as therapeutic drugs. At the same time, genetic engineering technology has brought revolutionary changes to drug production technology. In the past, some products that were difficult to produce, such as hormones, enzymes, antibodies and other bioactive substances, can be put into production with high quality and high yield through genetic engineering, and at the same time, the production cost is greatly reduced, which improves the medication level and quality of life of patients.

Genetic engineering technology has provided effective new means in the diagnosis, treatment and prevention of some diseases that cannot be effectively treated by traditional medicine, such as cancer, AIDS and genetic diseases, and has made some major breakthroughs. If oncogenes are found, early diagnosis of cancer and development of therapeutic drugs will become possible. With the development of molecular biology and gene recombination technology, we believe that in the near future, these diseases that seriously endanger human life will be effectively prevented and treated.