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Scapharca subcrenata-derived calcium binding protein ASP-3 with antitumor activity and structure thereof

An ASP-3, calcium binding protein technology, applied in the field of calcium binding protein and its preparation, can solve the problems of inclusion body formation, low efficiency and the like, and achieve the effect of improving anti-tumor activity

Pending Publication Date: 2022-01-18
于荣敏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Heterologous protein expression is a hot spot in modern medicine, industry, and basic research. Among them, the most commonly used E. coli heterologous expression system has the characteristics of clear genetic background, easy cultivation, and low cost. However, low efficiency often occurs during heterologous expression. , inclusion body formation, etc.

Method used

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  • Scapharca subcrenata-derived calcium binding protein ASP-3 with antitumor activity and structure thereof
  • Scapharca subcrenata-derived calcium binding protein ASP-3 with antitumor activity and structure thereof
  • Scapharca subcrenata-derived calcium binding protein ASP-3 with antitumor activity and structure thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the preparation method of recombinant clam (Arca subcrenata Lischke, Scapharca subcrenata) calcium-binding protein ASP-3

[0054] 1. Construction of the expression vector of the clam calcium-binding protein ASP-3 gene

[0055] 1.1 Total RNA extraction and cDNA library construction of cockles

[0056] The total RNA of clam was extracted by Trizol method (the clam was purchased from Huangsha Seafood Market, Guangzhou). Put the reagents required for RNA extraction, pipette gun, 1.5mL EP tube and enzyme-free tip into the ultra-clean bench and irradiate it with ultraviolet light for sterilization. Put the cockle samples wrapped in medicine spoon and tinfoil into liquid nitrogen to freeze; add alcohol to the mortar and pestle, sterilize and remove enzymes through high-temperature combustion, and then add liquid nitrogen to the mortar for cooling. The cockle sample was taken out, placed in a mortar, and ground to powder with a pestle. Take 2-3 scoops of sample...

Embodiment 2

[0113] Example 2 High expression of clam calcium-binding protein gene in E.coli BL21(pET-28a(+))

[0114] The pET-28a(+) recombinant plasmid containing the calcium binding protein gene constructed in Example 1 was transformed into the expression host E.coli BL21(DE3) to obtain the recombinant strain E.coli BL21(DE3). Spread on the LB medium plate containing 50mg / L kanamycin, and pick the positive transformants. Positive clones were cultured overnight at 37°C in 50 mL of LB liquid medium containing 50 mg / L kanamycin, and expanded to 500 mL of LB liquid medium containing 50 mg / L kanamycin at a ratio of 1:100. OD 600 Add IPTG inducer between 0.5-0.6 to a concentration of 0.5mM, and continue culturing at 37°C for 14h. Collect bacteria with LE Buffer (pH=7.4, 20mM Na 2 HPO 4 , 500mM NaCl) after resuspension, the bacterial cells were disrupted by ultrasonic treatment, centrifuged at 13000rpm for 30min at 4°C, and the supernatant was collected, which was the crude protein solutio...

Embodiment 3

[0117] Example 3 Screening of expression conditions in the preparation process of recombinant clam calcium-binding protein ASP-3

[0118] The specific steps are the same as those shown in Example 2. The screening scheme for expression conditions is shown in Table 9. The expression of ASP-3 under different concentrations of IPTG inducers, different temperatures, and different expression times was investigated, as shown in Table 9.

[0119] Table 9 ASP-3 crystallization condition optimization table

[0120]

[0121]

[0122] Conclusion: When no IPTG inducer is added, there is no expression of ASP-3 protein. When IPTG inducer is added to a concentration of 0.2, the expression of ASP-3 protein is small. When IPTG inducer is added to a concentration of 1.0mM, there are inclusion bodies When the IPTG inducer was added to a concentration of 0.5-0.8mM, the expression of ASP-3 protein was more and no inclusion body was formed, and when the final concentration of IPTG inducer was ...

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Abstract

The invention relates to a scapharca subcrenata-derived calcium binding protein ASP-3 with antitumor activity and a structure thereof, and belongs to the technical field of biology. The recombinant calcium binding protein contains 200 amino acids, and the total length of the corresponding coding gene nucleotide is 603bp. The protein acts on an EGFR target of HepG2 tumor cells to inhibit tumor growth. The scapharca subcrenata calcium binding protein can be used in the medicine and health care product industry, and can be further used for research of liver cancer molecular targeted therapeutic drugs, marine antitumor protein resources can be fully utilized, and considerable economic benefits can be generated by obtaining rich antitumor proteins.

Description

technical field [0001] The invention relates to a calcium-binding protein with anti-tumor activity derived from cockles and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] Liver cancer is a malignant tumor with high incidence and mortality, and it is also one of the malignant tumors with the worst prognosis. It is listed as the sixth most common cancer in the world, with about 900,000 newly diagnosed cases every year, and its mortality rate is the highest among all cancers. Ranked third, the 5-year survival rate is less than 20%. my country is also a country with a high incidence of liver cancer. In 2020, the number of new cases of liver cancer ranks fifth, and the number of deaths from liver cancer ranks second, which seriously endangers human life and health. At present, the treatment of liver cancer is a multidisciplinary comprehensive treatment, mainly including surgical resection of tumor tissue blocks, liver transpl...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/36C07K1/30C07K1/22C12N15/12C12N15/70
CPCC07K14/43504C12N15/70C12N2800/101
Inventor 于荣敏石慧宋丽艳
Owner 于荣敏
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