New immunoassay systems based on recombinant human lactoferrin

In this work, soluble and solid phase immunoreagents, including recombinant human lactoferrin (rhLF), a complex of rhLF with europium ions, rabbit antiserum to rhLF, anti-rhLF immunoglobulin purified by antigen-affinity chromatography and the conjugates of this immunoglobulin with an Eu3+ chelate or horseradish peroxidase have been obtained by a combination of biochemical and synthetic methods using rhLF as an initial compound. Biospecific interactions of the reagents in four immunochemical systems were assessed by measuring the enzyme activity or time-resolved fluorescence. The study resulted in the development of fast and precise immunoassays for biologically active rhLF in transgenic goat milk and in protein fractions obtained in the course of pure rhLF manufacture, as well as in pharmaceutical preparations and food additives.

Ameliorative effect of recombinant human lactoferrin on the premature ovarian failure in rats after cyclophosphamide treatments

This study investigated the effect of recombinant human lactoferrin (rhLF) on the premature ovarian failure (POF) of rats. After cyclophosphamide treatments, the POF rats were divided into the following groups: normal control group (NC), low-dose group (LD), medium-dose group (MD) and high-dose group (HD) of rhLF. After drug administrations, the ovarian indexes and hormonal levels were detected. After follicle number count, the proliferation and apoptosis were analyzed with the expressions of genes related with oogenesis, reactive oxygen species (ROS) production and apoptosis detected, followed by the calculation of oxidative stress and protein expressions. After 4-hydroperoxy cyclophosphamide (4-HC) treatments, the effect of rhLF on the proliferation, ROS production and gene expressions of primary rat granulosa cells (GCs) cultured in vitro were detected. After mating, the fertilities of POF rats were recorded. The result showed that the rhLF administrations up-regulated the ovarian index with the number of developing follicles increased and the decreases of hormonal levels conferred. The Ki-67 intensities of the MD and HD groups were up-regulated with the Tunnel intensities decreased. The rhLF treatments significantly promoted the expression of oogenesis, antioxidant and anti-apoptosis related genes. The expression of Bax and Caspase 3 were decreased with the expression of Bcl-2 up-regulated after rhLF administrations. The in vitro treatments of rhLF effectively conferred the toxicity of 4-HC on primary rat GCs. The fertility assessment showed the rhLF treatments up-regulated the offspring’s’ folliculogenesis, which confirmed the ameliorative role of rhLF on the POF damages via the inhibition of ROS production in GCs.

The concentration of recombinant human lactoferrin in the milk of transgenic goats of the third and fourth year of lactation

The concentration of recombinant human lactoferrin (rhLF) in the milk of 22 transgenic goats of different ages was studied: the average concentration of rhLF in the milk of goats-producers of the 4th year of lactation was 13.7% higher than that of goats of the 3rd year of lactation (6.07 and 5.24 g/l, respectively). It was found that the maximum level of recombinant glycoprotein was produced by the mammary gland of animals in the 2nd and 4th quarters of the studied year period. Protein’s concentration in milk measured in the 2nd and 4th quarters was 6.4 g/l and 6.33 g/l for producers of the third year of lactation and 8.14 g/l and 7.02 g/l for goats of the fourth year of lactation, respectively. The correlation between the season of the year and the content of the target protein in milk was detected (p <0.01), it was found that the most active rhLF production in goat milk occurred in late spring and winter periods. Variability in the content of the protein of interest in milk between individuals was noted. During the study a relatively low level of rhLF was established in animal №0747 - 0.11–2.52 g/l, while its maximum value was detected in the producer № 0403 - 4.52–9.06 g/l (more than 4 times difference). The range of individual variability in terms of rhLF content in the milk of goats of the third and the fourth lactation was 0.11–9.06 g/l. Milk of genetically modified goats contains 3 times more lactoferrin than human breast milk. Therefore, rhLF concentration testing in goat milk and tracking the dynamics of this indicator allows us to obtain data that can be used later for breeding pro-grams formation, milk production planning and also to improve the gene pool of transgenic animals.

DETERMINATION OF BIOLOGICALLY ACTIVE RECOMBINANT HUMAN LACTOFERRIN IN MILK OF TRANSGENIC GOATS AND PROTEIN FRACTIONS ISOLATED FROM MILK

Specific immunoreagents for the immunoassay technologies of recombinant human lactoferrin in food and biotechnological products have been obtained and characterized. This work was financially supported by the State program of scientific research «Chemical technologies and materials» for 2016 – 2020 years.

Recombinant human lactoferrin attenuates the progression of hepatosteatosis and hepatocellular death by regulating iron and lipid homeostasis in ob/ob mice

Lactoferrin was shown to possess antioxidant and anti-inflammatory properties and exert modulatory effects on lipid homeostasis and non-alcoholic fatty liver disease, but our understanding of its regulatory mechanisms is limited and inconsistent.