Construction of factor C from tachypleus tridentatus pronucleus and insect baculovirus recombinant expression vector

Abstract

The invention relates to the field of biotechnology, in particular to construction of a gene prokaryotic cell and insect cell expression plasmid of a factor C from tachypleus tridentatus. The gene sequence of the factor C from tachypleus tridentatus is shown by SEQ ID No.1 and consists of 3057bp; and the two ends of a primer for cloning respectively contain an NcoI enzyme digestion site and an NotI enzyme digestion site. The gene of the factor C from tachypleus tridentatus is cloned into a prokaryotic expression vector pET28a and a baculovirus expression vector pFASTBacHTA, and the DNA (deoxyribonucleic acid) sequencing proves that the sequence cloning is correct. The method successfully constructs a prokaryotic recombinant expression plasmid pET28a-FC and a baculovirus recombinant expression plasmid pFASTBacHTA-FC of the factor C from tachypleus tridentatus. The invention provides a new method for the heterogeneous expression of the factor C from tachypleus tridentatus, and also lays a foundation for the follow-up research and application of a large amount of expression of the factor C from tachypleus tridentatus in prokaryotic cells and insect cells.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the plasmid construction of the Chinese horseshoe crab factor C gene expression vector in prokaryotic cells and insect cells. Background technique [0002] Among clinical infusion reactions, pyrogen reaction is the most harmful and has the highest incidence rate. Endotoxin (lipopolysaccharide, LPS) is the most well-studied and most common biological pyrogen, and it is also a problem that major pharmaceutical companies must face, because even pg Trace levels of LPS can also cause severe pyrogen reactions in patients. Therefore, a sensitive and reliable endotoxin diagnostic technique is very necessary. [0003] The amoeba-like cells (amoebocytes) of sea crab have super sensitivity to LPS. When Gram-negative bacteria invade, it can produce a cascade agglutination reaction to the foreign invading bacteria through a series of enzymatic reactions, so that Invading microorganisms a...

AI Technical Summary

Problems solved by technology

However, there are only two kinds of LAL (Limulus amoebocytelysate) and TAL (Tachypleus Amebocyte Lysate) that can be used in the production of LAL (Limulus amebocyte Lysate) respectively. The two have the same reaction principle and efficacy, but there are also differences in physical and chemical properties. Differences, such as the optimum pH for reacting with endotoxin, etc. (6)

[0006]Although the limulus assay (LAL / TAL) has the advantages of high sensitivity (can detect femtogram-level endotoxin) and rapidity, due to the use of complex cell lysis As a reaction raw material, it also has insurmountable defects

For example, the problem of non-specific interference: In addition to reacting with endotoxin, Limulus reagent also reacts with (1-3)-β-D-glucan (1)

Therefore, it is easy to encounter non-specific interference of (1-3)-β-D-glucan in the detection process of Limulus reagent, resulting in false positive results. This is a big test for drugs with complex components, especially traditional Chinese medicine. challenge(7)

Another example is the issue of batch stability: Limulus reagent uses Limulus blood as the raw material for production. Due to differences in seasons and regions, batch differences in Limulus reagent become a common phenomenon.

Moreover, due to environmental pollution, overfishing and other reasons, the population of horseshoe crabs has decreased sharply in recent years and is close to extinction (8), which makes the raw materials for the production of Limulus reagents face the danger of increasing scarcity

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