Safety And Function Study On Recombinant Human Lactoferrin Expressed By Mammary Gland Bioreactor Of Transgenic Cows

ObjectivesTo study the safety of purified recombinant human lactoferrin (rhLf) expressed by mammary gland bioreactor of transgenic cows, and whether it displays functions of growth improvement, antibacteria and improving iron status.MethodsI Equivalence analysisEquivalence of rhLf and natural hLf in respect of structure, immunogenicity and biological activity was determined. The structure analysis consisted of molecular weight assay, N-terminal amino acid sequence analysis, glycosylation sites confirmation, and secondary structure determination. The immunogenicity was examined by comparing the immune response of rhLf and hLf with anti-hLf. Analysis of biological activity was focused on the capacity of iron binding and releasing.II Safety study1. Toxicity studyBioinformatics was conducted to determine the homology of rhLf and known toxic proteins or anti nutrients. Toxicity of short-term intake of rhLf was assessed in dose of 5g/kg bw/d for 7d and 35g/kg bw/d for 28d.2. Allergenicity studyBioinformatics was conducted to determine the homology of rhLf and known allergenic proteins. Digestion test of rhLf and hLf in simulate/biological gastric and intestinal fluid was performed and their stability against digestion was compared.III Function study1. Growth improvement effectThree groups of 14 male weaned Wistar rats were administered rhLf by gavage at doses of 0.375,0.75 or 2.25g/kg bw/d for 56d. Another group received distilled water as control. Body weight, body length and food utilization were recorded at regular intervals.2. Antibacterial effect in vitrorhLf at different concentrations was added into the culture medium of E. coli, Salmonella, P. aeruginosa, S. aureus and L. mono cy to genes, Growth status of each bacteria was measured at different time points.3. Effect of improving iron statusFemale weaning Wistar rats were fed with iron deficient diet for 30d. Animals with the level of Hb under 100g/L were chosen into the recovering stage and were randomly allocated into group A-F:model control,0.375,0.75 and 2.25g/kg bw rhLf supplement, 0.011g/kg bw ferrous lactate supplement, and mixture of 0.011g/kg bw ferrous lactate and 0.375g/kg bw rhLf supplement. Test substances were administered by gavage everyday for 8w. Indexes related were determined including general health condition, body weight, hematological parameters, FEP, SI, TIBC, TS, SF, SC and SZ.ResultsⅠEquivalence analysisThe molecular weight, N-terminal amino acid sequence, glycosylation sites and secondary structure of rhLf and hLf were all parallel. Identical immunogenicity was displayed. Both rhLf and hLf had the ability of iron binding and releasing.ⅡSafety study1. Toxicity study1.1 Bioinformatics analysisThere were 530 kinds of proteins homologous to rhLf in UniProt database, all of which belonged to the transferrin family.1.27-day oral toxicity study in ratsBody weight of all animals reduced after administering 5g/kg bw/d rhLf for 7d. The same results came out when the 7-day oral toxicity study was repeated on protein powder in dose of 3.3g/kg bw/d, which suggested the inappropriateness of assessing oral toxicity of protein by gavage in large dose. Two weeks after treatment ceased, there was no difference among rhLf group, protein powder group and control group in body weight, food consumption and utilization. The weight of organs, hematological, blood biochemical, and histopathological examinations showed no adverse effects in rhLf group.1.3 28-day feeding test in miceCompared with casein group, body weight of male mice in rhLf group increased in the 4th week. Levels of PLT, RDW, TP and ALB increased, while the relative weight of testicle reduced in male rhLf group. For female rhLf group, ALT, TRIG and the relative weight of kidney increased, while TP and CRE deceased. There was no significance for the other indexes.2. Allergenicity studyrhLf was homologous to bovine lactotransferrin, ovotransferrin and Amb p 5, three of which were known allergens. Stability against digestion in simulate/biological gastric and intestinal fluid was identical for rhLf and hLf. Both were easily digested in gastric fluid, but stayed stable for 60min in intestinal fluid.ⅢFunction study1. Growth improvement effectCompared with control group, rats of 2.25g/kg bw/d group got obvious improvement on physical parameters including body weight, body length and food utilization. Animals of 0.375 and 0.75g/kg bw/d group got slight improvement on physical parameters.2. Antibacterial effect in vitrorhLf inhibited the growth of E. coli, Salmonella, P. aeruginosa, S. aureus and L. monocytogenes in vitro, and the effect got stronger when concentration of rhLf increased within a certain range. Sensitivity of various strains to rhLf was different. L. monocytogenes was the most sensitive bacteria in the same condition.3. Effect of improving iron statusAfter 8 weeks of supplement, body weight, RBC, Hb, SI, TS and SF levels of ferrous lactate group and the mixture group were significantly higher than those of control, while levels of FEP, TIBC and SZ were lower. Indexes of 2.25g/kg rhLf group were also improved with a less degree. There was no obvious change in 0.375 or 0.75g/kg rhLf group compared with the control group except for larger amount of RBC and lower levels of FEP and SZ in 0.75g/kg rhLf group, and lower level of SZ in 0.375g/kg rhLf group. Regarding to the comparison between ferrous lactate group and the mixture group, SI, TS and SF levels of the mixture group were significantly higher than those of ferrous lactate group, with lower level of TIBC at the same time.ConclusionsⅠrhLf is equivalent to natural hLf in respect of structure, immunogenicity and biological activity.ⅡIt is demonstrated by the bioinformatics that there is no homology between rhLf and any known toxic protein or anti nutrient, while rhLf is found homologous to bovine lactotransferrin, ovotransferrin and Amb p 5 which are known allergens.ⅢOrally intake of rhLf in dose of up to 35g/kg bw/d for 28 d has no toxic effects on mice, which suggests high food safety of rhLf.ⅣStability against digestion in simulate/biological gastric and intestinal fluid is identical for rhLf and hLf, and neither has digestion resistance.ⅤrhLf expressed by mammary gland of transgenic cows is confirmed to display functions of improving growth in rats, antibacteria in vitro, and improving iron status in rats suffering IDA for the first time.