Constitutive Expression Of Porcine Lactoferrin And Lactoferrcin In Pichia Pastoris And Preliminary Exploration Of Biological Function

Lactoferrin is a natural biological iron-binding glycoprotein exists in ectocrine of mammals, it posesses muLtiple biological function. It is not only involved in iron transport, but also has the functions of anti-microbial, anti-oxidation, and reguLates the immune system. Lactoferricin is a kind of hydrolysis product from lactoferrin which has the more strong antimicrobial activity than lactoferrin. We calculated the antimicrobial activity gene of LFcinP on the basis of NCBI database’s publishing about amino acid sequence of lactoferrin form Cattle,Goat and Camel and the relatting documents’s information, analysising the sequence and physical and chemical properties. According to the aminoacid sequences of porcine lactoferrin and partiality codon of Pichia pastorsis, the LFcinP was designed and synthesized. the LFcinP gene was cloned Into the pGAPHaM vector to construct the recombinant expression vector pGAPHaM-LFcinP, the Bgl Ⅱ linearized plasmid was transformed into Pichia pastors GS115by electroporation. The transformants were identified by PCR, Under the control of the promoter GAP, the recombinant LFcinP protein with a molecuLar weight of5.4kDa was expressed in the supernatant, and the resuLts of96h SDS-PAGE indicates that recombinant protein account for35.9%of the total celluLar protein, theconcentration of expressed protein was48.5ug/mL;The natural lactoferrin was cloned951bp and named pLF-319, The pLF-319gene was cloned Into the pGAPHaM vector to construct the recombinant expression vector pGAPHaM-pLF-319, the Bgl Ⅱ linearized plasmid was transformed into Pichia pastors GS115by electroporation. The transformants were identified by PCR, Under the control of the promoter GAP, the recombinant pLF-319protein with a molecuLar weight of35kDa was expressed in the supernatant, and the resuLts of96h SDS-PAGE indicates that recombinant protein account for6.9%of the total celluLar protein, theconcentration of expressed protein was9.18ug/mL;The growth curve showed that the LFcinP and pLF-319had antibacterial abilities not only to gram-positibe bacterium but also to gram-negative bacterium. This study provides a basis for the lactoferricin applied in the animal production.Weaning BALB/c mice as model animals, the mice were divided into four groups, each group12mice, different groups were fed PBS, GS115supernatant, pGAPHaM-LFcinP/GS115supernatant and pGAPHaM-pLF-319supernatant, Effect of lactoferrin protein solution for21days,the detection of porcine lactoferrin and Lactoferrcin ability to improve weaning mice foodutilization, promoting the growth of weanling mice, and serum biochemical indicators. Daily food intake, water intake, weight and length of the mice in each group were measured, the resuLts show that the experimental group mice food utilization higher than that in the control group;length test showed that, taking the average length of Lactoferrin and Lactoferrcin increment was higher than4.5cm (PBS) and4.25cm (pastoris GS115); CHO1,GLU have no significant difference between the different groups, LFcinP group and pLF of-319significantly improve the level of TBIL, IBIL and D-BIL(P<0.05); AKP has no different between each group, comparing the PBS and GS115group with the LFcinP and pLF-319group,The Experimental group ALT activity was significantly decreased (P<0.05), LDH activity was significantly increased (P<0.05). These resuLts can be seen the reorganization of the porcine lactoferrin and Lactoferrcin can improve the digestive function of the weaning mice, enhanced immune function, has a role in promoting growth to weanling mice.