Method for finding protein interaction with baculovirus PCNA (Proliferating Cell Nuclear Antigen)

A baculovirus and protein technology, applied in the fields of genetic engineering and biology, can solve the problems of slow onset and lethal effect, high host specificity, and influence on the use of baculovirus.

Inactive Publication Date: 2014-04-09
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its high host specificity and slow onset and le...

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  • Method for finding protein interaction with baculovirus PCNA (Proliferating Cell Nuclear Antigen)
  • Method for finding protein interaction with baculovirus PCNA (Proliferating Cell Nuclear Antigen)
  • Method for finding protein interaction with baculovirus PCNA (Proliferating Cell Nuclear Antigen)

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Embodiment 1

[0026] Embodiment one: Cloning of PCNA gene of Autographa californica nuclear polyhedrosis virus

[0027] Primers were designed according to the PCNA gene sequence of AcMNPV (GenBank ID: 1403881). PCR primers are:

[0028] Forward: 5'-CGCGGATCCATGTTCGAAGCGGAATTT-3' (the underline is the BamHI restriction site)

[0029] Reverse: 5'-CCGCTCGAGGTGATGGTGATGGTGATG-3' (XhoⅠ restriction site is underlined)

[0030] PCNA fragments were specifically amplified with full gold TransStart FastPfu DNA Polymerase. PCR amplification conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 40 s, a total of 25 cycles. This was followed by an additional extension at 72°C for 8 min. The PCR product was subjected to agarose electrophoresis, and the 800bp fragment was cut out, and the recovered amplified fragment was purified with the gel extraction kit of Shanghai Jierui. The target fragment was connected to the p...

Embodiment 2

[0032] Embodiment two: expression and purification of PCNA protein

[0033] The PCNA-30a recombinant plasmid was transformed into Escherichia coli BL21 competent cells. Pick the correctly identified target clone and inoculate it in LB medium, culture it in a shaker overnight at 37°C, transfer it to fresh LB medium the next day, and add directly to the bacterial solution when the OD600 of the bacterial solution is 0.6 IPTG for induction. After 8 hours, the induced cells were collected and processed, and the treated cell proteins were subjected to 12% SDS-PAGE electrophoresis, and the proteins after gel separation were transferred to PVDF membranes, and mouse-derived anti-His monoclonal The antibody was immunoreacted, and the Western blot detection result of His-PCNA with 6 X His tag was as follows: figure 1 shown.

[0034] Use 1L Escherichia coli containing PCNA-30a fusion protein expression vector for large-scale induction, centrifuge at 4000g for 15 minutes after induction...

Embodiment 3

[0035] Example 3: Preparation of PCNA coupled CNBr-Activated Sepharose 4B column

[0036]Dissolve 5 mg / mL PCNA in coupling buffer (0.1M NaCO3 containing 0.5M NaCl, pH 8.3). 1mM HCl swells CNBr-Activated Sepharose 4B lyophilized powder. The coupling solution containing PCNA and the swollen CNBr-Activated Sepharose 4B were mixed for 2 hours; pH 8.0, 1M ethanolamine was added to eliminate residual active groups, and the column was packed. Wash the coupling medium alternately 3 times with 0.1 M acetate buffer (pH 4.5) containing 0.5 M NaCl and coupling buffer.

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Abstract

The invention discloses a method for finding a protein interaction with baculovirus PCNA (Proliferating Cell Nuclear Antigen). The method is characterized by comprising the following steps of cloning full-length DNA of an autographa californica nuclear polyhedrosis virus (AcMNPV) PCNA gene by utilizing the means such as molecular cloning, and DNA sequencing, purifying the full-length DNA, coupling the full-length DNA to an affinity column, and fining the interaction protein by adopting the mass spectrography. The method can be used for finding and functionally studying interaction protein of the baculovirus PCNA.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biotechnology, in particular to a protein interacting with Proliferating Cell Nuclear Antigen (PCNA) of Autographa californica Nuclear Polyhedrosis Virus (AcMNPV). technical background [0002] Baculoviruses are a group of pathogenic microorganisms that specifically parasitize arthropods. Among the numerous members of baculoviruses, Autographa californica nuclear polyhedrosis virus (AcMNPV) is the most studied and utilized. The host domain of baculoviruses is narrow and restricted to a few species of arthropods, mainly insects. The outstanding advantages of baculovirus for biological control are: it is safe for humans and animals, does not harm natural enemies, is highly pathogenic to hosts, has a long-lasting aftereffect of killing insects, does not pollute the environment, and has aftereffects. However, its high host specificity and slow onset and lethality also affect the use of bacul...

Claims

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Application Information

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IPC IPC(8): G01N27/62C12N15/34C12N15/70C07K14/01C07K1/22C07K1/18
Inventor 陈慧卿周亚竟唐琦李梅黄国平李国辉麦维军张春霞
Owner JIANGSU UNIV
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