US 12,246,045 B2
Method for preparing a transformed, Salmo salar interferon gamma (IFNG)-producing Lactococcus lactis bacterium
Mario Cesar Gerardo Tello Reyes, Santiago (CL); Alvaro Eugenio Santibañez Vargas, Santiago (CL); Mick Philippe Parra Mardonez, Santiago (CL); Diego Enrique Paine Cabrera, Santiago (CL); Claudia Andrea Zapata Rojas, Santiago (CL); and Andrea Del Pilar Garces Fernandez, Santiago (CL)
Assigned to CONSORICO TECNOLOGICO DE SANIDAD ACUICOLA S.A., Santiago (CL); and UNIVERSIDAD DE SANTIAGO DE CHILE, Santiago (CL)
Filed by CONSORCIO TECNOLOGICO DE SANIDAD ACUICOLA S.A., Santiago (CL); and UNIVERSIDAD DE SANTIAGO DE CHILE, Santiago (CL)
Filed on Sep. 14, 2023, as Appl. No. 18/467,280.
Application 18/467,280 is a division of application No. 16/764,356, previously published as PCT/CL2018/050021, filed on Apr. 20, 2018.
Claims priority of application No. 2897-2017 (CL), filed on Nov. 14, 2017.
Prior Publication US 2024/0131088 A1, Apr. 25, 2024
Int. Cl. A61K 35/744 (2015.01); A23K 10/18 (2016.01); A23K 50/80 (2016.01); A61K 9/00 (2006.01); A61K 38/21 (2006.01); C12N 1/20 (2006.01); C12N 15/74 (2006.01); C12R 1/46 (2006.01)
CPC A61K 35/744 (2013.01) [A23K 10/18 (2016.05); A23K 50/80 (2016.05); A61K 9/0053 (2013.01); A61K 38/217 (2013.01); C12N 1/205 (2021.05); C12N 15/746 (2013.01); A23V 2400/231 (2023.08); C12N 2800/101 (2013.01); C12R 2001/46 (2021.05)] 1 Claim
OG exemplary drawing
 
1. A method of preparing transformed Lactococcus lactis (L. lactis) bacteria to produce interferon gamma (IFNγ) comprising steps of:
a) digesting a plasmid containing IFNγ with restriction enzymes NcoI and XbaI to form a digested product,
b) purifying the digested product to form a first purified product;
c) digesting a plasmid pNZ8149 with restriction enzymes NcoI and XbaI;
d) purifying the plasmid pNZ8149 from an agarose gel to form a second purified product;
e) ligating the first and second purified products from steps b) and d) with ligase to form a ligated plasmid;
f) dialyzing the ligated plasmid; and
g) transforming electrocompetent L. lactis bacteria NZ3900 with the ligated plasmid to form a transformed L. lactis bacteria having the accession number RGM 2416 comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is a DNA construct P1-USP45-IFNγ-GGG-6×His, wherein the DNA construct is within the plasmid pNZ8149, and
wherein
P1 is a L. lactis constitutive expression promoter;
Usp45 is a secretion signal;
IFNγ is the mature mRNA coding sequence for Salmo salar interferon gamma that is codon optimized for expression in L. lactis;
GGG is a sequence coding for three glycines; and
6×His is a sequence coding for 6 terminal histidines.