Using Cell Surface Display Technology Combined With Intestinal Repair To Reduce Pb²⁺ Residues In Grass Carp ?Catenopharyngodon Idella?

Lead is a heavy metal that naturally exists deep underground.In the past thousands of years,due to human activities,the content of lead in the environment has risen sharply.Lead in soil and atmosphere will enter the water environment through the precipitation and erosion of rainwater,pollute the water system and accumulate in the tissues of aquatic animals,and then transfer to the human through the food chain.Lead will accumulate in different tissues of the body over time and is not easily degraded,it has adverse effects on the nervous system,immunity,blood,reproductive system,and gastrointestinal tract.Because the main ways of human exposure to lead are contaminated water,soil and food,so blocking lead absorption in the gastrointestinal tract is a reasonable preventive strategy.With the development of molecular display technology,the display of heavy metal ion-binding proteins on the surface of engineering bacteria is considered as a promising and sustainable way to remove heavy metal pollution in the environment,and has become one of the research hotpots in the field of environment.However,few studies have been conducted on the adsorption of heavy metals by engineering bacteria in biological bodies.This topic use cell display technology,select the protein of PbBD with specific adsorption of Pb²⁺and anchor it on the surface of BL21?DE3?by INP,to study the effect of engineering bacteria E.coli PbBD on the reduction of lead content in various tissues of grass carp exposed to lead and the repair of intestinal flora.Grass carps were randomly divided into four groups?control,Pb,Pb+PbBD and PbBD group?.Pb²⁺was added into the fish diet?5mg/kg grass carps?and fed for 7weeks.The engineering bacteria group add feed containing E.coli PbBD?10⁸CFU/g diet?was provided twice a day.At the end of the experiment,samples of tissues and fecal were collected,lead levels and biochemical markers were detected,and changes in intestinal villi and mucus,as well as changes in intestinal microbial structure were analyzed.The results showed that E.coli PbBD had a strong ability and specificity to absorb Pb²⁺from the solution in vitro.In vivo,1).E.coli PbBD can successfully survive and colonize the gut of grass carp.2).E.coli PbBD can adsorb Pb²⁺in the intestine and expel it out with feces,effectively reducing the lead accumulation in the muscle,bone,gill,viscera and other tissues of grass carp,and reducing the lead level in the edible part of the fish.3).Pb+PbBD group alleviated the oxidative stress induced by exposure to Pb²⁺,reduced the levels of MDA and ROS,and increased the levels of GSH-px,CAT and T-SOD.4).E.coli PbBD alleviates the damage to intestinal cells and mucus caused by lead.5).E.coli PbBD can adjust the proportion of pathogenic bacterias such as Fusobacteria,Shigella,Bacteroides and beneficial bacteria such as Cetobacterium,Gemmobacter and Akkermansia in the intestinal tract caused by lead toxicity,improve the intestinal flora disorder caused by Pb²⁺,protect the intestinal health of the host and maintain the intestinal flora homeostasis.6).There was no adverse effect on the growth,biochemical indicators and intestinal flora of grass carp,when E.coli PbBD was added only.In summary,the addition of E.coli PbBD showed some effects in reducing the toxicity of Pb²⁺,and use of E.coli PbBD only has no adverse effect on the health of grass carp,it is feasible to add E.coli PbBD in fish food to reduce the accumulation of lead.Therefore,the engineered bacteria that can absorb Pb²⁺constructed in this study can be used as the"lead removal tool"for grass carp to prevent the safety problems caused by Pb²⁺pollution in grass carp farming.