Method for preparing human DNA polymerase delta by using bombyx mori bioreactor

Abstract

The invention relates to a method for preparing human DNA polymerase delta by using a bombyx mori bioreactor. The method comprises the following specific steps of: respectively constructing four subunits of the human DNA polymerase delta onto a donor plasmid; carrying out transposition on Tn7 transposons in transformed escherichia coli DH10Bac / BmNPV (Bombyx mori Nuclear Polyhedrosis Virus) and BmNPV DNA and screening out clones with correct subunits; and then preparing recombinant viruses of the four subunits assembled by different combinations in BmN cells; infecting host bombyx mori; performing the expression and assembly of a multi-subunit protein complex in bombys mori; and collecting the body liquid of infected larvas or homogenizing entirely, and finally separating and purifying to obtain the target product. By using the method disclosed in the invention, the DNA polymerase multi-subunit protein complex which has high specific activity and is assembled correctly can be prepared efficiently in large scale with low cost. The method can be widely applied to the field of researches on some important diseases, especially on cancers, and the basic research on DNA replication and injury repair.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and biotechnology, in particular to a method for preparing human DNA polymerase delta multi-subunit protein complexes using a silkworm bioreactor. DNA polymerase for in vitro studies of DNA replication and damage repair. Background technique [0002] "Faithful" DNA replication plays a key role in the regulation of the eukaryotic cell cycle, and regulatory disturbances occur at a certain stage of the cell cycle, for example, in deciding when to start DNA replication, or whether it can be successfully completed in S-phase The replication of DNA, etc., is an important cause of various human diseases, especially cancer diseases. Therefore, accurate DNA replication and damage repair play an extremely important role in eukaryotes to maintain their chromosome stability and prevent genetic variation and cancer disease. [0003] The eukaryotic DNA polymerase Pol δ (Polymerase δ, Pol δ) is the...

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Problems solved by technology

It was extremely difficult to isolate and purify the polymerase Pol δ from animal tissues in the early stage, requiring at least 8 separation columns, which took several months, and only about 33 micrograms of protein could be obtained from 1 kg of calf pancreas (Lee, M.Y., et al, Biochemistry, 1984, 23: 1906-1913), and it is easily inactivated, so large-scale enzymatic function and protein structure analysis cannot be carried out. The difficulty in the separation and purification of natural Pol δ is that the research field of eukaryotic polymerase lags behind other systems DNA polymerization One of the main reasons for enzyme research

Although the AcMNPV insect baculovirus-Bac-to-Bac expression system was applied in 2002, Pol δ protein complexes with different subunit assembly combinations were obtained by co-infecting Sf9 insect cells by preparing viruses of each subunit (Xie, B., et al. al, Biochemistry, 2002, 41: 13133-13142; Podust, V.N., et al., J Biol Chem, 2002, 277: 3894-3901), but due to its low expression level, it is impossible to obtain a large amount of high-purity enzyme, and the enzyme The preparation process involves the expensive medium and fetal bovine serum required for insect cell culture, which makes the enzyme preparation cost high and severely restricts the in-depth research in the field of DNA polymerase Pol δ

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