Study On The Difference Of Venoms From Wild And Captive Cobra(Naja Atra)

In recent years, the edible and medicinal value of Naja atra has been gradually paid more attentions, and many factors lead to the decrease in the number of wild N.atra, so, in order to meet the needs of the market and protection of wild N. atra,artificial feed and no hibernation technology were used to feed N. atra fast. Therefore,N. atra was breeded with a growing scale in our country. But so far, a few of reports have pointed out that the venom from the wild and the captive snakes have some difference in some extent. There is no systematic analysis and identification of their specific differences in any aspects. Furthermore, snake venom is mainly composed of dozens of proteins(enzyme) or a mixture of polypeptides components(or "ammunition"), these proteins or peptides are expressed in the venom gland cells. Many studies reported have showed that through millions of years of accelerated evolution,toxin genes encoding these toxins were recruited to venom gland cells, and caused the difference of proteins of venom. This study plans to find the specific differences between the captive and the wild cobra N. atra by comparing and analyzing the components of venom, enzyme activities in venom, the level of transcription of toxin genes, pharmacological activities of venom, and the influence of different food to the captive cobra venom. This study provides bases for supporting and guiding for producing venoms and antivenoms of N. atra. After a series of experiments, the results or conclusions are obtained as follows:(1) Results of SDS-PAGE assaying:Under the reduction condition,SDS-PAGE indicated that the number of the protein bands(95 kD) of the venom from the wild cobra, is more than with captive cobra,and the content of proteins(55 kD) of the wild cobra more higher than the captive; Under the non-reduction condition;The content of high molecular weight proteins(170 kD) of the venom of the wild cobra more higher than the captive; results of RP-HPLC assaying: the chromatogram peak area of the venom of wild cobra which is more larger than that in captive.(2) Results of enzyme activity assaying: The activity of PLA2 was measured by the lecithin plate method, the activity is expressed by the size of the transparent zone, Theaverage diameters of the transparent zone of 30 μg of wild or captive cobra venom were16.45 mm and 18.38 mm, respectively; The activity of hyaluronidase was measured by the hyaluronic acid plate method, the activity is expressed by the size of the transparent zone, the average diameters of the transparent zone of 30 μg wild or captive cobra venom were 12.81 mm and 11.57 mm, respectively; the L-AAO activity was defined as the absorbance at 490 nm when leucine was taken as substrates, and the L-AAO activities of 10 μg of wild or captive cobra venom were 0.341 and 0.397, respectively;the alkaline phosphomonoesterase activity is defined as the absorbance at 405 nm when PNPP was taken as substrates, and the alkaline phosphomonoesterase activities of 30 μg of wild or captive cobra venom were 0.199 and 0.184, respectively; the acetylcholinesterase activity was defined as the absorbance at 405 nm absorbance when acetylcholine iodide was taken as substrates, and the acetylcholinesterase activities of30 μg of wild or captive cobra venom were 0.297 and 0.413, respectively; the proteinase activity was defined as the absorbance at 630 nm when Azo-casein was taken as substrates, the proteinase activities of 30 μg of wild or captive cobra venom were 0.104 and 0.106, respectively, which indicated no significant difference between them; when AMP was taken as substrates, The 5’-nucleotidase activity of 20 μg wild or captive cobra venom was 82.17 U and 75.83 U, respectively.(3) Results of pharmacological activity analysis: The swelling degree of mice paw by subcutaneous injection with wild or captive cobra venom were 3.6 mm and 3.86 mm,respectively. The analgesic time was prolonged to 7.5 s and 6.86 s, respectively, after injection of 8 μg of wild or captive cobra venom into the tail vein of mice. The dosages of LD50 of wild or captive cobra venom, by intraperitoneal injection of venom, were0.79 μg/g and 0.81 μg/g, respectively. But the death rate of the wild cobra venom is faster than the captive cobra venom.(4) According to transcriptome results: Compared to wild cobra venom gland, the mRNA expressive quantity of 5’-NT, L-AAO, protease and AchE in captive cobra venom gland showed a significant up-regulation trend, and mRNA expressive quantity of PLA2 and neurotoxins were down-regulated.(5) When the different food were provided to the captive cobra, the growth rates of the cobra were different. The growth rate of the cobra fed with mice is slower, but the growth rate of the cobra fed with artificial food is faster. Under the non-reduction condition, SDS-PAGE indicated that the number of the protein bands of the venom from the captive cobra fed with mice, is more than with artificial food. The content ofhigh molecular weight proteins of the venom of the young captive cobra fed with mice is higher than that of the young captive cobra fed with artificial food; after the cobra was fed for four months, the activities of PLA2 and protease from the venom of cobra fed artificial food much were higher than that in captive cobra fed with mice; the activity of hyaluronidase from the venom of cobra fed with artificial food was much lower than that in captive cobra fed with mice.