Study On Liposomes Of DNA Photolyase From Antarctic Ice Microalgae

DNA photolyase is a kind of enzyme that is found in Antarctic ice microalgae Chlamydomonas sp.ICE-L,which can repair the DNA damage caused by ultraviolet light and restore its normal spatial structure.In this paper,we used DNA photolyase as the target drug,which was found in Chlamydomonas sp.ICE-Lof Antarctic ice microalgae,and prepared freeze-dried preparations in the form of liposome,so as to make it play the role of sunscreen.This paper is divided into four parts:1.The process was studied on the extraction,separation and purification of DNA photolyase from Antarctic ice microalgae.The results showed that the effective conditions for obtaining the effective protein were 5000 rpm centrifugal 20 min,ultrasonic broken 20 min in ice bath.SDS-PAGE expression was detected in the expressed and purified proteins,and the bands of the target proteins appeared near66 KD.The addition of mannitol had a protective effect on the activity of DNA repair enzyme.2.The preparation technology of DNA photolyase liposome from Antarctic ice microalgae was studied.The results showed that the liposome was prepared by reverse evaporation method,and the solid soybean lecithin and cholesterol was used as the membrane material,and the mixed solvent of chloroform-ether was organicphase.The optimal formulation of liposomes was determined by single factor experiment and orthogonal experiment with the encapsulation efficiency as an index.The optimal formulation was that: the ratio of membrane to material was 3: 1,the ratio of oil to water was 4: 1,the ratio of drug to membrane was 1:10,and the ultrasonic time was 6 min.Hydration was carried out in phosphate buffer at pH = 7.0for 30 min and the hydration temperature was 30 ℃.The average encapsulation efficiency of the final product prepared at the optimal formulation was 44.13±2.90%.3.The freeze-drying process of liposomes was studied.Appearance morphology,entrapment efficiency,particle size,redispersibility of freeze-drying process were evaluation index.The optimum preparation process was as follows: the liposomes were pre-frozen at-80℃for 12 h,freezed for 24 h,and trehalose was the freeze-dried protective agent.The encapsulation efficiency of lyophilized liposomes was 42.89 ±1.44%.Under the condition of low temperature,the appearance were stable and no agglomeration,and the partical size and entrapment efficiency were not changed and the activity was stable.4.The quality standard detection system was establish with the appearance,entrapment efficiency,particle size and Zeta potential of liposomes.The morphology of the DNA photolyase liposomes was round or oval,and the particle size of liposomes and lyophilized preparations were 490.9±2.3nm and 591.3±1.6nm,and the Zeta potential was in the range of-30 mV to-60 mV.The content of liposomal preparations was determined by UV analysis.The results showed that the specificity,precision,stability and repeatability of UV analysis were good.The recoveries of liposomes were 99.44% ~ 100.22% and the RSD was 0.80% ~ 1.55%,which proved that the method was accurate and reliable.This subject had completed the DNA photolyase lyophilized liposome,the method of preparation was simple and feasible.The lyophilized preparation quality standard meet the requirements of Chinese Pharmacopoeia 2015 edition,it provided a reliable theoretical basis for the application of DNA photolyase in the daily sun.