Genetic Engineering Sericin Hydrogel For The Oral Delivery Of Recombinant Human Lactoferrin(rhLF)for Enhancing Immunity In Mice

Silkworm（Bombyx mori.）has function of silk secretion,which is the core of important economic insects at present.With the development of molecular biotechnology,people are no longer satisfied with mulberry-silk only used for silk production,but also hope to develop new uses of silk through new research technology to serve human beings,such as using silk protein regeneration to prepare drug delivery system and improve the utilization efficiency of drugs;changing feeding methods to produce conductive silk;using genetic methods to produce silk with mechanical properties comparable to spider silk;using proteomics to search medical protein and so on.Due to their unique physiological and biochemical characteristics,silkworm is received much attention.The silk gland of silkworm has strong ability of silk protein synthesis and secretion,and the genetic background is clear with the completion of the silkworm genome trilogy,indicating that silkworm has the potential as bioreactor.Researchers have made some achievements in this field,a variety of exogenous proteins were successful expression in the silk gland of silkworm,and exogenous proteins were secreted to cocoons with silk protein,the exogenous proteins"inlays"in the silk.So far,the growth factor is the most successfully expressed by silk gland bioreactor of silkworm.Expanding the type of protein is of great significance for improving the value of silk and the application of the silk gland bioreactor.Silk protein has the characteristics of preparing biomaterials.Many studies have shown that biomaterials made from regenerated silk fibroin and sericin can be used for tissue engineering repair or as drug carrier.Therefore,combine the silk gland as bioreactor with the advantages of silk protein of preparing biomaterials is of great significance to improve the added value of silk.Besides,it can expand the application scope of silkworm and helpful for the silkworm industry development.What’s more,it also rich resource of the production of recombinant protein and provides new directions for the development of bioengineering in the future.Based on the above considerations,the following research works were carried out in this study:1)Expression an immune protein,recombinant human lactoferrin（rhLF）,in the middle silk gland of silkworm expression system.To identify the successful amount of rhLF and established a new silkworm strain with stable heredity according to the highest expression level and to investigate the economic characteristics of rhLF silkworm strain.2)Optimization of purification procedure to obtain purified rhLF.Determination the purity of rhLF and calculation of purification yield.Secondary structure and glycosylation modification of rhLF were also detected.3)Detection of the anti-inflammatory and antibacterial activities of rhLF in vitro.4)Fabrication an rhLF-loaded sericin hydrogel using rhLF-carrying silk cocoons produced by a transgenic silkworm strain.Detection the physicochemical properties of hydrogels of secondary structure,morphology,rhLF release characteristics from hydrogel and stability of rhLF hydrogel.5)The toxicity of rhLF hydrogels in vivo and in vitro.6)The activity of rhLF hydrogel for enhancing immunosuppressed mice immunity.7)pharmacokinetics of rhLF hydrogel in mice.The main results of this study are as follows:1.Generation of rhLF transgenic silkworm strainHuman lactoferrin（hLF）is a multifunctional glycoprotein,which is an important part of innate immunity.The host produces non-specific defense mechanism immediately or within hours after contact with antigen.Neutrophils in blood or inflammatory tissue can release a lot of LF with body infection.LF competitive combined with LPS could reduce the damage of LPS to the body.LF is widely used in market.For medicine,it can prevent premature delivery or treat chronic inflammation of the elderly;for food,it can be added to infant formula milk powder as a nutritional supplement;for beauty,it can be added to cosmetics.The market demand for LF is very strong.We used the middle silk gland of silkworm expression system to express rhLF.We first looked up the hLF coding sequence from NCBI,optimized and synthesized rhLF gene according to silkworm codon preference,constructed the expression vector of middle silk gland of silkworm based on piggyBac.ACSI enzyme was used to detect whether the vector was constructed successfully.Then,the expression vector was microinjected into the eggs（G0 generation）and the hatchling eggs were raised.12positive moth circles were obtained by fluorescent screening of G1 generation eggs,pupae and moths,with a positive rate of 27.9%.The results of SDS-PAGE showed that the molecular weight of the distinct protein bands was between 70 to 100 kDa,which is in accordance with the theoretical size of the rhLF（⁸0 kDa）.Further immunoblot blot analysis showed that these distinct protein bands could react with the anti-human lactoferrin antibody.The positive transgenic silkworm strain 34 with the highest expression of rhLF was maintained to generate a stable transgenic strain.Insertion of the rhLF expression cassette was mediated by the piggyBac transposon into a“TTAA”preference site on Chr.8,nscaf 2827:8517795-8518010 of the silkworm genome.We further investigated the economic shape of rhLF silkworm.There was no significant difference of the total cocoon quantity,cocoon layer quantity and cocoon layer rate between rhLF and WT silkworm strain.2.Purification,structure and activity detection of rhLFThe rhLF sericin was extracted from rhLF cocoons,and detected by SDS-PAGE and Western blot.After a complete extraction,a total 12.07 mg rhLF could be recovered from 1 g cocoons from the transgenic silkworm No.34 strain.A 6×his flag was added to rhLF gene,so we can purify the rhLF by Ni-charged His-binding column.Finally,9.24 mg rhLF with a recovery of 76.55%could be purified from 1g of cocoons from the transgenic silkworm.Furthermore,the purity of rhLF from five independent processing steps was 95.45%with a variability coefficient of 0.82%.CD Pro showed that rhLF and rhLF-STD contain considerablely similarα-helix,β-fold,β-angle,and irregular curl structures.The result of N-glycosylation detection showed that eight types of N-glycans which dominated by the GlcNAc（4）Man（3）（61.15%）and the GlcNAc（3）Man（3）（17.98%）were identified at the three typical N-glycosylation sites of the rhLF.Biological activities assays showed the significant evidence that the purified rhLF could relief the lipopolysaccharide（LPS）-induced cell inflammation in RAW264.7 cells and exhibit potent antibacterial bioactivities against the Escherichia coli（E.coli）and Bacillus subtilis.3.Preparation and characterization of rhLF sericin hydrogelsSericins from WT and rhLF silks were extracted using 8 M urea buffer to fabricate sericin hydrogels.Sericin,with or without recombinant human lactoferrin（rhLF）,was steadily retained in aqueous condition in 8 M urea solution and gradually transformed into hydrogels along with urea removal by dialysis.Using this method,we have prepared WT and two kinds of rhLF hydrogels,namely rhLF-sh-L and rhLF-sh-H sericin hydrogels.FTIR characterizations according to amide I peak separation method revealed four absorption peaks which appeared at 1614 cm^-1,1640 cm^-1,1663 cm^-1,and1687 cm^-1,representingβ-sheet,random coil,α-helix,andβ-turn,respectively.The highestβ-sheet content was 46.80%,followed by random coil（27.86%）.Subsequently,the external and internal structures of freeze-dried hydrogels were analyzed via SEM.The hydrogel showed a uniform morphology and regular porous microstructure.Release characteristics of rhLF from hydrogels were detected under different pH conditions（pH4.0,7.0,and 9.0）.Release behaviors of rhLF in different pH environments were significantly different,as faster release was observed under acidic conditions than neutral and alkaline conditions.31.45%of total rhLF in sericin hydrogels was released under acidic conditions within 240 minutes.After 8 weeks of storage at 4,25,and37℃,rhLF retentions in the hydrogels were 83.81%,77.95%,and 77.15%,respectively,indicating that low temperature was helpful to the preservation of rhLF hydrogel.4.Toxicity of rhLF hydrogel in vitro and in vivoThe toxicity of the rhLF sericin hydrogel was first evaluated in vitro.HIH/3T3cells co-cultured with WT-sh and rhLF-sh-L hydrogel did not cause significant cell death or morphological abnormalities.Before further testing the toxicity of sericin hydrogel in vivo,mice were starved treat for 12h,then administered the large dose of WT and rhLF sericin hydrogel by gavage,and mice observed for 14 days.During the observation period,no toxic signs were found in the appearance of mice.The weight of the mice increased normally without obvious hair loss or abnormal nasal secretions,and all the mice survived the observational period.The autopsies showed that the heart,liver,spleen,lung,and kidney of the mice had no abnormal color or shape.Furthermore,there was no significant difference in the routine blood,partial liver,and kidney function tests between healthy and hydrogel-treated mice.In summary,these results show that the fabricated rhLF sericin hydrogel was non-cytotoxic.5.Therapeutic efficacy of rhLF hydrogel on the CTX-induced immunosuppression mice modelChemotherapy is one of the main means of cancer treatment.The use of chemotherapy drugs often brings serious side effects to patients,such as atrophy of immune organs,nausea,vomiting,hair loss and so on.A CTX-induced immunosuppression mouse model was constructed.Then,Saline,WT-sh,rhLF-sh-L,rhLF-sh-H,and rhLF-STD were used to gavage immunosuppressed mice daily.The rhLF-STD was used as a positive control.Before the end of the experiment,a variety indexes of immune related were measured.The thymus and spleen of mice were remodeled after treatment of Cyclophosphamide（CTX）immunosuppressed mice with the rhLF sericin hydrogel,which also enhanced the expression of CD3 molecules in the spleen of the mice.Combinations based on the phagocytic index,delayed-type hypersensitivity response（DTH）tests,and forced swimming tests revealed that oral administration of rhLF sericin hydrogel helped to clear foreign bodies,maintain stable environment,and prolong the swimming time of mice.The total mRNA of spleen in mice was extracted.We further analyzed cytokine expression in CTX mice from different groups and observed that rhLF hydrogel promoted the expression of immunoregulatory factors CD3,IL21,IL2,and IL18 in a dose-dependent manner.Intestinal flora is considered as another immune organ.16S rRNA analysis was used to investigate gut microbiome in feces.Venn plots were used to compare the uniqueness of intestinal microflora in treatment and CK groups.The differences between groups S,WT-sh,rhLF-sh-L,rhLF-sh-H,and rhLF-STD and the CK group were 2.54%,1.95%,1.09%,1.4%,and 1.24%,respectively.6.Pharmacokinetics of rhLF in vivoThe rhLF-sh-L（170μg/mL,0.3 mL）or the aqueous rhLF-STD solution（170μg/mL,0.3 mL）was fed to two groups of mice through gavage.ELISA showed that the maximum plasma concentration of rhLF in mice from rhLF-sh-L and rhLF-STD solution groups were 70 and 90 min,respectively.Notably,the plasma concentrations of rhLF in rhLF-sh-L-treated mice were significantly higher than that in rhLF-STD solution-treated mice at time points ranging from 70-150 minutes after drug administration.Accordingly,the bioavailability of rhLF-sh-L was calculated as 38%higher than that of the aqueous rhLF-STD solution.Near-infrared fluorescence（NIRF）imaging was used to evaluate the bio-distribution of rhLF-sh-L hydrogel in vivo,strong NIRF signals were observed in the stomachs and intestines of mice after treatment with rhLF-sh-L hydrogel for 12 hThese results suggest that sericin hydrogel can protect rhLF from complex digestive system and prolong the residence time in the gastrointestinal tract,thereby contributing to the absorption of rhLF by the body to enhance the immunity of immunosuppressed mice.